Compositions and methods for silencing vegf-a expression

ABSTRACT

The disclosure relates to double-stranded ribonucleic acid (dsRNA) compositions targeting VEGF-A, and methods of using such dsRNA compositions to alter (e.g., inhibit) expression of VEGF-A.

RELATED APPLICATIONS

This application claims priority to U.S. provisional application No. 62/972,519, filed on Feb. 10, 2020, U.S. provisional application No. 63/055,627, filed on Jul. 23, 2020, and U.S. provisional application No. 63/140,714, filed on Jan. 22, 2021. The entire contents of the foregoing applications are hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 4, 2021, is named A2038-7236WO_SL.txt and is 1,327,255 bytes in size.

FIELD OF THE DISCLOSURE

The disclosure relates to the specific inhibition of the expression of the VEGF-A.

BACKGROUND

Vascular eye diseases are the leading cause of vision loss in today's aging population, including exudative age-related macular degeneration (exudative AMD), retinal vein occlusion (RVO), retinopathy of prematurity (ROP), diabetic retinopathy (DR), and diabetic macular edema (DME). Several of these ocular disorders are associated with pathological angiogenesis. The release of vascular endothelial growth factors (VEGFs) contributes to increased vascular permeability and inappropriate new vessel growth in the eye. New treatments for angiogenic ocular disorders are needed.

SUMMARY

The present disclosure describes methods and iRNA compositions for modulating the expression of VEGF-A. In certain embodiments, expression of VEGF-A is reduced or inhibited using a VEGF-A-specific iRNA. Such inhibition can be useful in treating disorders related to VEGF-A expression, such as ocular disorders (e.g., age-related macular degeneration (AMD), macular edema following retinal vein occlusion (MEfRVO) or central retinal vein occlusion (CVO), retinopathy of prematurity (ROP), diabetic macular edema (DME), and diabetic retinopathy (DR)).

Accordingly, described herein are compositions and methods that effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of VEGF-A, such as in a cell or in a subject (e.g., in a mammal, such as a human subject). Also described are compositions and methods for treating a disorder related to expression of VEGF-A, such as an angiogenic ocular disorder (e.g., AMD, RVO, MEfRVO, CVO, ROP, DME, mCNV, and DR)).

The iRNAs (e.g., dsRNAs) included in the compositions featured herein include an RNA strand (the antisense strand) having a region, e.g., a region that is 30 nucleotides or less, generally 19-24 nucleotides in length, that is substantially complementary to at least part of an mRNA transcript of VEGF-A (e.g., a human VEGF-A) (also referred to herein as a “VEGF-A-specific iRNA”). In some embodiments, the VEGF-A mRNA transcript is a human VEGF-A mRNA transcript, e.g., SEQ ID NO: 1 herein.

In some embodiments, the iRNA (e.g., dsRNA) described herein comprises an antisense strand having a region that is substantially complementary to a region of a human VEGF-A mRNA. In some embodiments, the human VEGF-A mRNA has the sequence NM_001171623.1 (SEQ ID NO: 1). The sequence of NM_001171623.1 is also herein incorporated by reference in its entirety. The reverse complement of SEQ ID NO: 1 is provided as SEQ ID NO: 2 herein.

In some aspects, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of vascular endothelial growth factor A (VEGF-A), wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of a coding strand of human VEGF-A and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of a non-coding strand of human VEGF-A such that the sense strand is complementary to the at least 15 contiguous nucleotides in the antisense strand.

In some aspects, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of VEGF-A, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 15 contiguous nucleotides in the antisense strand.

In some aspects, the present disclosure provides a human cell or tissue comprising a reduced level of VEGF-A mRNA or a level of VEGF-A protein as compared to an otherwise similar untreated cell or tissue, wherein optionally the cell or tissue is not genetically engineered (e.g., wherein the cell or tissue comprises one or more naturally arising mutations, e.g., VEGF-A), wherein optionally the level is reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the human cell or tissue is a retinal pigment epithelium (RPE), a retinal tissue, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, a photoreceptor cell, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel.

The present disclosure also provides, in some aspects, a cell containing the dsRNA agent described herein.

In another aspect, provided herein is a human ocular cell, e.g., (an RPE cell, a retinal cell, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, or a photoreceptor cell) comprising a reduced level of VEGF-A mRNA or a level of VEGF-A protein as compared to an otherwise similar untreated cell. In some embodiments, the level is reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

In some aspects, the present disclosure also provides a pharmaceutical composition for inhibiting expression of a gene encoding VEGF-A, comprising a dsRNA agent described herein.

The present disclosure also provides, in some aspects, a method of inhibiting expression of VEGF-A in a cell, the method comprising:

(a) contacting the cell with the dsRNA agent described herein, or a pharmaceutical composition described herein; and

(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of VEGF-A, thereby inhibiting expression of the VEGF-A in the cell.

The present disclosure also provides, in some aspects, a method of inhibiting expression of VEGF-A in a cell, the method comprising:

(a) contacting the cell with the dsRNA agent described herein, or a pharmaceutical composition described herein; and

(b) maintaining the cell produced in step (a) for a time sufficient to reduce levels of VEGF-A mRNA, VEGF-A protein, or both of VEGF-A mRNA and protein, thereby inhibiting expression of the VEGF-A in the cell.

The present disclosure also provides, in some aspects, a method of inhibiting expression of VEGF-A in an ocular cell or tissue, the method comprising:

(a) contacting the cell or tissue with a dsRNA agent that binds VEGF-A; and

(b) maintaining the cell or tissue produced in step (a) for a time sufficient to reduce levels of VEGF-A mRNA, VEGF-A protein, or both of VEGF-A mRNA and protein, thereby inhibiting expression of VEGF-A in the cell or tissue.

The present disclosure also provides, in some aspects, a method of treating a subject diagnosed with VEGF-A-associated disorder comprising administering to the subject a therapeutically effective amount of the dsRNA agent described herein or a pharmaceutical composition described herein, thereby treating the disorder.

In any of the aspects herein, e.g., the compositions and methods above, any of the embodiments herein (e.g., below) may apply.

In some embodiments, the coding strand of human VEGF-A has the sequence of SEQ ID NO: 1. In some embodiments, the non-coding strand of human VEGF-A has the sequence of SEQ ID NO: 2.

In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 17 contiguous nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 19 contiguous nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 21 contiguous nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

In some embodiments, the portion of the sense strand is a portion within nucleotides 1855-1875, 1858-1878, 2178-2198, 2181-2201, 2944-2964, 2946-2966, 2952-2972, 3361-3381, or 3362-3382 of SEQ ID NO: 1. In some embodiments, the portion of the sense strand is a portion corresponding to SEQ ID NO: 4200, 4201, 4202, 4203, 4204, 4205, 4206, 4207, 4208, 4209, 4210, or 4211.

In some embodiments, the portion of the sense strand is a portion within a sense strand in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B.

In some embodiments, the portion of the antisense strand is a portion within an antisense strand in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B.

In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B that corresponds to the antisense sequence.

In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B that corresponds to the antisense sequence.

In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A 8B, 10A, 10B, 12, 13, 14, 18A and 18B that corresponds to the antisense sequence.

In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A 8B, 10A, 10B, 12, 13, 14, 18A and 18B that corresponds to the antisense sequence.

In some embodiments, the sense strand of the dsRNA agent is at least 23 nucleotides in length, e.g., 23-30 nucleotides in length.

In some embodiments, the portion of the sense strand is a portion within a sense strand from a duplex chosen from AD-1020574 (CGACAGAACAGUCCUUAAUCA (SEQ ID NO: 4200)), AD-901094 (CAGAACAGUCCUUAAUCCAGA (SEQ ID NO: 4201)), AD-1020575 (CAGAACAGUCCUUAAUCCAGA (SEQ ID NO: 4202)), AD-901100 (AACAGUGCUAAUGUUAUUGGA (SEQ ID NO: 4203)), AD-901101 (AGUGCUAAUGUUAUUGGUGUA (SEQ ID NO: 4204)), AD-901113 (GAGAAAGUGUUUUAUAUACGA (SEQ ID NO: 4205)), AD-901123 (AAAAUAGACAUUGCUAUUCUA (SEQ ID NO: 4206)), AD-901124 (AAAUAGACAUUGCUAUUCUGA (SEQ ID NO: 4207)), AD-901158 (GAAAGUGUUUUAUAUACGGUA (SEQ ID NO: 4208)), AD-901159 (GUUUUAUAUACGGUACUUAUA (SEQ ID NO: 4209)), AD-1020573 (AGUGCUAATGTUAUUGGUGUA (SEQ ID NO: 4210)), or AD-1023143 (AAAAUAGACATUGCUAUUCUA (SEQ ID NO: 4211)). In some embodiments, the portion is a portion of a corresponding chemically modified sequence provided in Tables 2A, 3A, 4A, or Table 18A.

In some embodiments, the portion of the sense strand is a sense strand chosen from the sense strands of AD-1020574 (CGACAGAACAGUCCUUAAUCA (SEQ ID NO: 4200)), AD-901094 (CAGAACAGUCCUUAAUCCAGA (SEQ ID NO: 4201)), AD-1020575 (CAGAACAGUCCUUAAUCCAGA (SEQ ID NO: 4202)), AD-901100 (AACAGUGCUAAUGUUAUUGGA (SEQ ID NO: 4203)), AD-901101 (AGUGCUAAUGUUAUUGGUGUA (SEQ ID NO: 4204)), AD-901113 (GAGAAAGUGUUUUAUAUACGA (SEQ ID NO: 4205)), AD-901123 (AAAAUAGACAUUGCUAUUCUA (SEQ ID NO: 4206)), AD-901124 (AAAUAGACAUUGCUAUUCUGA (SEQ ID NO: 4207)), AD-901158 (GAAAGUGUUUUAUAUACGGUA (SEQ ID NO: 4208)), AD-901159 (GUUUUAUAUACGGUACUUAUA (SEQ ID NO: 4209)), AD-1020573 (AGUGCUAATGTUAUUGGUGUA (SEQ ID NO: 4210)), or AD-1023143 (AAAAUAGACATUGCUAUUCUA (SEQ ID NO: 4211)). In some embodiments, the portion is a portion of a corresponding chemically modified sequence provided in Tables 2A, 3A, 4A, or Table 18A.

In some embodiments, the portion of the sense strand is a portion within a sense strand from a duplex chosen from AD-953374 (SEQ ID NO: 813), AD-953504 (SEQ ID NO: 1297), AD-953481 (SEQ ID NO: 1298), AD-953351 (SEQ ID NO: 800), AD-901356 (SEQ ID NO: 261), AD-953344 (SEQ ID NO: 787), AD-901355 (SEQ ID NO: 262), AD-953410 (SEQ ID NO: 845), AD-953363 (SEQ ID NO: 779), AD-953411 (SEQ ID NO: 844), AD-953350 (SEQ ID NO: 784), or AD-953375 (SEQ ID NO: 790). In some embodiments, the portion is a portion of a corresponding chemically modified sequence provided in Tables 2A, 3A, 4A, or Table 18A.

In some embodiments, the portion of the sense strand is a sense strand chosen from the sense strands of AD-953374 (SEQ ID NO: 813), AD-953504 (SEQ ID NO: 1297), AD-953481 (SEQ ID NO: 1298), AD-953351 (SEQ ID NO: 800), AD-901356 (SEQ ID NO: 261), AD-953344 (SEQ ID NO: 787), AD-901355 (SEQ ID NO: 262), AD-953410 (SEQ ID NO: 845), AD-953363 (SEQ ID NO: 779), AD-953411 (SEQ ID NO: 844), AD-953350 (SEQ ID NO: 784), or AD-953375 (SEQ ID NO: 790). In some embodiments, the portion is a portion of a corresponding chemically modified sequence provided in Tables 2A, 3A, 4A, or Table 18A.

In some embodiments, the portion of the antisense strand is a portion within an antisense strand from a duplex chosen from AD-1020574 (UGAUUAAGGACUGUUCUGUCGAU (SEQ ID NO: 4212)), AD-901094 (UCUGGAUUAAGGACUGUUCUGUC (SEQ ID NO: 4213)), AD-1020575 (UCUGGATUAAGGACUGUUCUGUC (SEQ ID NO: 4214)), AD-901100 (UCCAAUAACAUUAGCACUGUUAA (SEQ ID NO: 4215)), AD-901101 (UACACCAAUAACAUUAGCACUGU (SEQ ID NO: 4216)), AD-901113 (UCGUAUAUAAAACACUUUCUCUU (SEQ ID NO: 4217)), AD-901123 (UAGAAUAGCAAUGUCUAUUUUAU (SEQ ID NO: 4218)), AD-901124 (UCAGAAUAGCAAUGUCUAUUUUA (SEQ ID NO: 4219)), AD-901158 (UACCGUAUAUAAAACACUUUCUC (SEQ ID NO: 4220)), AD-901159 (UAUAAGUACCGUAUAUAAAACAC (SEQ ID NO: 4221)), AD-1020573 (UACACCAAUAACATUAGCACUGU (SEQ ID NO: 4222)), or AD-1023143 (UAGAAUAGCAATGTCTAUUUUAU (SEQ ID NO: 4223)). In some embodiments, the portion is a portion of a corresponding chemically modified sequence provided in Tables 2A, 3A, 4A, or Table 18A.

In some embodiments, the portion of the antisense strand is an antisense strand chosen from the antisense strands of AD-1020574 (UGAUUAAGGACUGUUCUGUCGAU (SEQ ID NO: 4212)), AD-901094 (UCUGGAUUAAGGACUGUUCUGUC (SEQ ID NO: 4213)), AD-1020575 (UCUGGATUAAGGACUGUUCUGUC (SEQ ID NO: 4214)), AD-901100 (UCCAAUAACAUUAGCACUGUUAA (SEQ ID NO: 4215)), AD-901101 (UACACCAAUAACAUUAGCACUGU (SEQ ID NO: 4216)), AD-901113 (UCGUAUAUAAAACACUUUCUCUU (SEQ ID NO: 4217)), AD-901123 (UAGAAUAGCAAUGUCUAUUUUAU (SEQ ID NO: 4218)), AD-901124 (UCAGAAUAGCAAUGUCUAUUUUA (SEQ ID NO: 4219)), AD-901158 (UACCGUAUAUAAAACACUUUCUC (SEQ ID NO: 4220)), AD-901159 (UAUAAGUACCGUAUAUAAAACAC (SEQ ID NO: 4221)), AD-1020573 (UACACCAAUAACATUAGCACUGU (SEQ ID NO: 4222)), or AD-1023143 (UAGAAUAGCAATGTCTAUUUUAU (SEQ ID NO: 4223)). In some embodiments, the portion is a portion of a corresponding chemically modified sequence provided in Tables 2A, 3A, 4A, or Table 18A.

In some embodiments, the portion of the antisense strand is a portion within an antisense strand from a duplex chosen from AD-953374 (SEQ ID NO: 943), AD-953504 (SEQ ID NO: 1427), AD-953481 (SEQ ID NO: 1428), AD-953351 (SEQ ID NO: 930), AD-901356 (SEQ ID NO: 390), AD-953344 (SEQ ID NO: 917), AD-901355 (SEQ ID NO: 391), AD-953410 (SEQ ID NO: 975), AD-953363 (SEQ ID NO: 909), AD-953411 (SEQ ID NO: 974), AD-953350 (SEQ ID NO: 914), or AD-953375 (SEQ ID NO: 920). In some embodiments, the portion is a portion of a corresponding chemically modified sequence provided in Tables 2A, 3A, 4A, or Table 18A.

In some embodiments, the portion of the antisense strand is an antisense strand chosen from the antisense strands of AD-953374 (SEQ ID NO: 943), AD-953504 (SEQ ID NO: 1427), AD-953481 (SEQ ID NO: 1428), AD-953351 (SEQ ID NO: 930), AD-901356 (SEQ ID NO: 390), AD-953344 (SEQ ID NO: 917), AD-901355 (SEQ ID NO: 391), AD-953410 (SEQ ID NO: 975), AD-953363 (SEQ ID NO: 909), AD-953411 (SEQ ID NO: 974), AD-953350 (SEQ ID NO: 914), or AD-953375 (SEQ ID NO: 920). In some embodiments, the portion is a portion of a corresponding chemically modified sequence provided in Tables 2A, 3A, 4A, or Table 18A.

In some embodiments, the sense strand and the antisense strand of the dsRNA agent comprise nucleotide sequences of the paired sense strand and antisense strand of a duplex selected from AD-1020574 (SEQ ID NO: 4200 and 4212), AD-901094 (SEQ ID NO: 4201 and 4213), AD-1020575 (SEQ ID NO: 4202 and 4214), AD-901100 (SEQ ID NO: 4203 and 4215), AD-901101 (SEQ ID NO: 4204 and 4216), AD-901113 (SEQ ID NO: 4205 and 4217), AD-901123 (SEQ ID NO: 4206 and 4218), AD-901124 (SEQ ID NO: 4207 and 4219), AD-901158 (SEQ ID NO: 4208 and 4220), AD-901159 (SEQ ID NO: 4209 and 4221), AD-1020573 (SEQ ID NO: 4210 and 4222), or AD-1023143 (SEQ ID NO: 4211 and 4223). In some embodiments, the sense strand and antisense strand comprises the corresponding chemically modified sense sequence and antisense sequence provided in Tables 2A, 3A, 4A, or Table 18A.

In some embodiments, the sense strand and the antisense strand of the dsRNA agent comprise nucleotide sequences of the paired sense strand and antisense strand of a duplex selected from AD-953374 (SEQ ID NO: 813 and 943), AD-953504 (SEQ ID NO: 1297 and 1427), AD-953481 (SEQ ID NO: 1298 and 1428), AD-953351 (SEQ ID NO: 800 and 930), AD-901356 (SEQ ID NO: 261 and 390), AD-953344 (SEQ ID NO: 787 and 917), AD-901355 (SEQ ID NO: 262 and 391), AD-953410 (SEQ ID NO: 845 and 975), AD-953363 (SEQ ID NO: 779 and 909), AD-953411 (SEQ ID NO: 844 and 974), AD-953350 (SEQ ID NO: 784 and 914), or AD-953375 (SEQ ID NO: 790 and 920). In some embodiments, the sense strand and antisense strand comprises the corresponding chemically modified sense sequence and antisense sequence provided in Tables 2A, 3A, 4A, or Table 18A.

In some embodiments, at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties. In some embodiments, the lipophilic moiety is conjugated to one or more positions in the double stranded region of the dsRNA agent. In some embodiments, the lipophilic moiety is conjugated via a linker or carrier. In some embodiments, lipophilicity of the lipophilic moiety, measured by log Kow, exceeds 0. In some embodiments, In some embodiments, the hydrophobicity of the double-stranded RNAi agent, measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent, exceeds 0.2. In some embodiments, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

In some embodiments, the dsRNA agent comprises at least one modified nucleotide. In some embodiments, no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand are unmodified nucleotides. In some embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In some embodiments, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′-phosphate mimic, a glycol modified nucleotide, and a 2-O—(N-methylacetamide) modified nucleotide; and combinations thereof. In some embodiments, no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand include modifications other than 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA).

In some embodiments, the dsRNA comprises a non-nucleotide spacer (wherein optionally the non-nucleotide spacer comprises a C3-C6 alkyl) between two of the contiguous nucleotides of the sense strand or between two of the contiguous nucleotides of the antisense strand.

In some embodiments, each strand is no more than 30 nucleotides in length. In some embodiments, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In some embodiments, at least one strand comprises a 3′ overhang of at least 2 nucleotides. In some embodiments, at least one strand comprises a 3′ overhang of 2 nucleotides.

In some embodiments, the double stranded region is 15-30 nucleotide pairs in length. In some embodiments, the double stranded region is 17-23 nucleotide pairs in length. In some embodiments, the double stranded region is 17-25 nucleotide pairs in length. In some embodiments, the double stranded region is 23-27 nucleotide pairs in length. In some embodiments, the double stranded region is 19-21 nucleotide pairs in length. In some embodiments, the double stranded region is 21-23 nucleotide pairs in length. In some embodiments, each strand has 19-30 nucleotides. In some embodiments, each strand has 19-23 nucleotides. In some embodiments, each strand has 21-23 nucleotides.

In some embodiments, the agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand. In some embodiments, the strand is the antisense strand. In some embodiments, the strand is the sense strand.

In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand. In some embodiments, the strand is the antisense strand. In some embodiments, the strand is the sense strand.

In some embodiments, each of the 5′- and 3′-terminus of one strand comprises a phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the strand is the antisense strand.

In some embodiments, the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

In some embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

In some embodiments, one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand. In some embodiments, the one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand via a linker or carrier.

In some embodiments, the internal positions include all positions except the terminal two positions from each end of the at least one strand. In some embodiments, the internal positions include all positions except the terminal three positions from each end of the at least one strand. In some embodiments, the internal positions exclude a cleavage site region of the sense strand. In some embodiments, the internal positions include all positions except positions 9-12, counting from the 5′-end of the sense strand. In some embodiments, the internal positions include all positions except positions 11-13, counting from the 3′-end of the sense strand. In some embodiments, the internal positions exclude a cleavage site region of the antisense strand. In some embodiments, the internal positions include all positions except positions 12-14, counting from the 5′-end of the antisense strand. In some embodiments, the internal positions include all positions except positions 11-13 on the sense strand, counting from the 3′-end, and positions 12-14 on the antisense strand, counting from the 5′-end.

In some embodiments, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5′end of each strand. In some embodiments, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5′-end of each strand.

In some embodiments, the positions in the double stranded region exclude a cleavage site region of the sense strand.

In some embodiments, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand. In some embodiments, the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 21, position 20, or position 15 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 20 or position 15 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 16 of the antisense strand. In some embodiments, the lipophilic moiety is conjugated to position 6, counting from the 5′-end of the sense strand.

In some embodiments, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound. In some embodiments, the lipophilic moiety is selected from the group consisting of lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

In some embodiments, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) or the double stranded region. In some embodiments, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.

In some embodiments, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.

In some embodiments, the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.

In some embodiments, the lipophilic moiety or targeting ligand is conjugated via a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

In some embodiments, the 3′ end of the sense strand is protected via an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

In some embodiments, the dsRNA agent further comprises a targeting ligand, e.g., a ligand that targets an ocular tissue or a liver tissue. In some embodiments, the ocular tissue is a retinal pigment epithelium (RPE) or choroid tissue, e.g., a choroid vessel.

In some embodiments, the ligand is conjugated to the sense strand. In some embodiments, the ligand is conjugated to the 3′ end or the 5′ end of the sense strand. In some embodiments, the ligand is conjugated to the 3′ end of the sense strand.

In some embodiments, the ligand comprises N-acetylgalactosamine (GalNAc). In some embodiments, the targeting ligand comprises one or more GalNAc conjugates or one or more GalNAc derivatives. In some embodiments, the ligand is one or more GalNAc conjugates or one or more GalNAc derivatives are attached through a monovalent linker, or a bivalent, trivalent, or tetravalent branched linker. In some embodiments, the ligand is

In some embodiments, the dsRNA agent is conjugated to the ligand as shown in the following schematic

wherein X is O or S. In some embodiments, the X is O.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first internucleotide linkage at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, second and third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In some embodiments, the dsRNA agent further comprises a phosphate or phosphate mimic at the 5′-end of the antisense strand. In some embodiments, the phosphate mimic is a 5′-vinyl phosphonate (VP).

In some embodiments, a cell described herein, e.g., a human cell, was produced by a process comprising contacting a human cell with the dsRNA agent described herein.

In some embodiments, a pharmaceutical composition described herein comprises the dsRNA agent and a lipid formulation.

In some embodiments (e.g., embodiments of the methods described herein), the cell is within a subject. In some embodiments, the subject is a human. In some embodiments, the level of VEGF-A mRNA is inhibited by at least 50%. In some embodiments, the level of VEGF-A protein is inhibited by at least 50%. In some embodiments, the expression of VEGF-A is inhibited by at least 50%. In some embodiments, inhibiting expression of VEGF-A decreases the VEGF-A protein level in a biological sample (e.g., an aqueous ocular fluid sample) from the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, inhibiting expression of VEGF-A gene decreases the VEGF-A mRNA level in a biological sample (e.g., an aqueous ocular fluid sample) from the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

In some embodiments, the subject has been diagnosed with a VEGF-A-associated disorder. In some embodiments, the subject meets at least one diagnostic criterion for a VEGF-A-associated disorder. In some embodiments, the VEGF-A associated disorder is wet age-related macular degeneration (wet AMD), diabetic retinopathy (DR), diabetic macular edema (DME), retinal vein occlusion (RVO), macular edema following retinal vein occlusion (MEfRVO), retinopathy of prematurity (ROP), or myopic choroidal neovascularization (mCNV). In some embodiments, the VEGF-A associated disorder is macular edema, e.g., diabetic macular edema.

In some embodiments, the ocular cell or tissue is RPE, a retinal cell, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, a photoreceptor cell, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel.

In some embodiments, the VEGF-A-associated disorder is an angiogenic ocular disorder. In some embodiments, the angiogenic ocular disorder is caused by or associated with the growth or proliferation of blood vessels. In some embodiments, the angiogenic ocular disorder is caused by or associated with ocular neovascularization. In some embodiments, the angiogenic ocular disorder is AMD, DR, DME, RVO, MEfRVO, ROP, or mCNV.

In some embodiments, treating comprises amelioration of at least one sign or symptom of the disorder. In some embodiments, the at least one sign or symptom includes a measure of one or more of angiogenesis, choroidal neovascularization, ocular inflammation, visual acuity, or presence, level, or activity of VEGF-A (e.g., VEGF-A gene, VEGF-A mRNA, or VEGF-A protein).

In some embodiments, a level of the VEGF-A that is higher than a reference level is indicative that the subject has an angiogenic ocular disorder. In some embodiments, treating comprises prevention of progression of the disorder. In some embodiments, the treating comprises one or more of (a) inhibiting angiogenesis; (b) inhibiting or reducing the expression or activity of VEGF-A; (c) inhibiting choroidal neovascularization; (d) inhibiting growth of new blood vessels in the choriocapillaris; (e) reducing retinal thickness; (f) increasing visual acuity; or (g) reducing intraocular inflammation.

In some embodiments, the treating results in at least a 30% mean reduction from baseline of VEGF-A mRNA in the retina, RPE, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel. In some embodiments, the treating results in at least a 60% mean reduction from baseline of VEGF-A mRNA in the retina, RPE, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel. In some embodiments, the treating results in at least a 90% mean reduction from baseline of VEGF-A mRNA in the retina, RPE, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel.

In some embodiments, after treatment the subject experiences at least an 8-week duration of knockdown following a single dose of dsRNA as assessed by VEGF-A protein in the retina. In some embodiments, treating results in at least a 12-week duration of knockdown following a single dose of dsRNA as assessed by VEGF-A protein in the retina. In some embodiments, treating results in at least a 16-week duration of knockdown following a single dose of dsRNA as assessed by VEGF-A protein in the retina.

In some embodiments, the subject is human.

In some embodiments, the dsRNA agent is administered at a dose of about 0.01 mg/kg to about 50 mg/kg.

In some embodiments, the dsRNA agent is administered to the subject intraocularly. In some embodiments, the intraocular administration comprises intravitreal administration, e.g., intravitreal injection; transscleral administration, e.g., transscleral injection; subconjunctival administration, e.g., subconjunctival injection; retrobulbar administration, e.g., retrobulbar injection; intracameral administration, e.g., intracameral injection, or subretinal administration, e.g., subretinal injection.

In some embodiments, the dsRNA agent is administered to the subject intravenously. In some embodiments, the dsRNA agent is administered to the subject topically.

In some embodiments, a method described herein further comprises measuring a level of VEGF-A (e.g., VEGF-A gene, VEGF-A mRNA, or VEGF-A protein) in the subject. In some embodiments, measuring the level of VEGF-A in the subject comprises measuring the level of VEGF-A protein in a biological sample from the subject (e.g., an aqueous ocular fluid sample). In some embodiments, a method described herein further comprises performing a blood test, an imaging test, or an aqueous ocular fluid biopsy (e.g., an aqueous humor tap).

In some embodiments, a method described herein further measuring level of VEGF-A (e.g., VEGF-A gene, VEGF-A mRNA, or VEGF-A protein) in the subject is performed prior to treatment with the dsRNA agent or the pharmaceutical composition. In some embodiments, upon determination that a subject has a level of VEGF-A that is greater than a reference level, the dsRNA agent or the pharmaceutical composition is administered to the subject. In some embodiments, measuring level of VEGF-A in the subject is performed after treatment with the dsRNA agent or the pharmaceutical composition.

In some embodiments, a method described herein further comprises treating the subject with a therapy suitable for treatment or prevention of a VEGF-A-associated disorder, e.g., wherein the therapy comprises photodynamic therapy, photocoagulation therapy, or vitrectomy. In some embodiments, a method described herein further comprises administering to the subject an additional agent suitable for treatment or prevention of a VEGF-A-associated disorder. In some embodiments, the additional agent comprises a steroid, a non-steroidal anti-inflammatory agent, or an anti-VEGF-A agent.

In some embodiments, the anti-VEGF-A agent comprises a fusion protein or an anti-VEGF-A antibody or antigen-binding fragment thereof (e.g., an anti-VEGF-A antibody molecule).

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

The details of various embodiments of the disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and the drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the sequences and chemistry of the exemplary VEGF-A siRNAs including AD-64228 (SEQ ID NO: 4162 and 4163), AD-953374 (SEQ ID NO: 553 and 683), AD-953504 (SEQ ID NO: 1037 and 1167), AD-953336 (SEQ ID NO: 518 and 648), AD-953337 (SEQ ID NO: 522 and 652), AD-901376 (SEQ ID NO: 4157 and 131), AD-953364 (SEQ ID NO: 567 and 697). FIG. 1B depicts the sequences and chemistry of the exemplary VEGF-A siRNAs including AD-953340 (SEQ ID NO: 517 and 647), AD-953351 (SEQ ID NO: 540 and 670), AD-953342 (SEQ ID NO: 523 and 653), AD-953308 (SEQ ID NO: 579 and 709), AD-953344 (SEQ ID NO: 527 and 657), AD-953339 (SEQ ID NO: 528 and 658), and AD-953363 (SEQ ID NO: 519 and 649). For each siRNA, “F” is the “2′-fluoro” modification, OMe is a methoxy group, GNA refers to a glycol nucleic acid, “DNA” refers to a DNA base, 2-C16 refers to the targeting ligand, and PS refers to the phosphorothioate linkage.

FIG. 2 is a graph depicting the percent VEGF-A message remaining normalized to PBS in mice on day 14 post-treatment with the exemplary duplexes indicated on the X-axis (from left to right: PBS control, naïve control, AAV positive control (AD-64228), AD-901376.2, AD-953308.2, AD-953336.2, AD-953337.2, AD-953339.2, AD-953340.2, AD-953342.2, AD-953344.2, AD-953351.2, AD-953363.2, AD-953364.2, AD-953374.2, AD-953504.2).

FIG. 3A depicts the sequences and chemistry of the exemplary VEGF-A siRNAs including AD-901349 (SEQ ID NO: 4156 and 130), AD-953481 (SEQ ID NO: 1038 and 1168), AD-901356 (SEQ ID NO: 3 and 132), AD-901355 (SEQ ID NO: 4 and 133), AD-953365 (SEQ ID NO: 552 and 682), AD-953410 (SEQ ID NO: 585 and 715), AD-953411 (SEQ ID NO: 584 and 714). FIG. 3B depicts the sequences and chemistry of the exemplary VEGF-A siRNAs including AD-953338 (SEQ ID NO: 520 and 650), AD-953350 (SEQ ID NO: 524 and 654), AD-953375 (SEQ ID NO: 530 and 660), AD-953341 (SEQ ID NO: 532 and 662), AD-953370 (SEQ ID NO: 533 and 663), AD-953386 (SEQ ID NO: 541 and 671), AD-64958 (SEQ ID NO: 5003 and 5004). For each siRNA, “F” is the “2′-fluoro” modification, OMe is a methoxy group, GNA refers to a glycol nucleic acid, 2-C16 refers to the targeting ligand, and PS refers to the phosphorothioate linkage.

FIG. 4 is a graph depicting the percent VEGF-A message remaining normalized to PBS in mice on day 14 post-treatment with the exemplary duplexes indicated on the X-axis (from left to right: PBS control, naïve control, AD-901349.1, AD-953481.1, AD-901356.1, AD-901355.1, AD-953365.1, AD-953410.1, AD-953411.1, AD-953338.1, AD-953350.1, AD-953375.1, AD-953341.1, AD-953370.1, AD-953386.1, and AD-64958 (ELF8 TTR control).

FIG. 5A depicts the sequences and chemistry of the exemplary VEGF-A siRNAs including AD-1397050 (SEQ ID NO: 5005 and 3936), AD-1397051 (SEQ ID NO: 5006 and 3918), AD-1397052 (SEQ ID NO: 10 and 3957), AD-1397053 (SEQ ID NO: 5007 and 3924), AD-1397054 (SEQ ID NO: 5008 and 2640), AD-1397055 (SEQ ID NO: 5009 and 2775). FIG. 5B depicts the sequences and chemistry of the exemplary VEGF-A siRNAs including AD-1397056 (SEQ ID NO: 5010 and 2776), AD-1397058 (SEQ ID NO: 5011 and 3953), AD-1397059 (SEQ ID NO: 5012 and 3889), AD-1397060 (SEQ ID NO: 5013 and 3902), AD-1397061 (SEQ ID NO: 5014 and 3932), and AD-1397062 (SEQ ID NO: 5015 and 3944). FIG. 5C depicts the sequences and chemistry of the exemplary VEGF-A siRNAs including AD-1397064 (SEQ ID NO: 5016 and 3938), AD-1397065 (SEQ ID NO: 5017 and 3965), AD-1397066 (SEQ ID NO: 5018 and 3962), AD-1397067 (SEQ ID NO: 5019 and 3971), AD-1397068 (SEQ ID NO: 1044 and 3901), AD-1397069 (SEQ ID NO: 5020 and 3928), and AD-64958 (SEQ ID NO: 5003 and 5004). For each siRNA, “F” is the “2′-fluoro” modification, OMe is a methoxy group, GNA refers to a glycol nucleic acid, “(A2p)” refers to adenosine 2′-phosphate, “(C2p)” refers to cytosine 2′-phosphate, “(U2p)” refers to uracil 2′-phosphate, “DNA” refers to a DNA base, 2-C16 refers to the targeting ligand, and PS refers to the phosphorothioate linkage.

FIG. 6 is a graph depicting the percent VEGF-A message remaining normalized to PBS in mice on day 14 post-treatment with the exemplary duplexes indicated on the X-axis (from left to right: PBS control, naïve control, AD-1397050.2, AD-1397051.2, AD-1397052.2, AD-1397053.2, AD-1397054.2, AD-1397055.2, AD-1397056.2, AD-1397058.2, AD-1397059.2, AD-1397060.2, AD-1397061.2, AD-1397062.2, AD-1397064.2, AD-1397065.2, AD-1397066.2, AD-1397067.2, AD-1397068.2, AD-1397069.2, and AD-64958.100.

DETAILED DESCRIPTION

iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Described herein are iRNAs and methods of using them for modulating (e.g., inhibiting) the expression of VEGF-A. Also provided are compositions and methods for treatment of disorders related to VEGF-A expression, such as an angiogenic ocular disorder (e.g., wet age-related macular degeneration (wet AMD), diabetic retinopathy (DR), diabetic macular edema (DME), retinal vein occlusion (RVO), macular edema following retinal vein occlusion (MEfRVO), retinopathy of prematurity (ROP), or myopic choroidal neovascularization (mCNV)).

Human VEGF-A is a dimeric glycoprotein of approximately 40 kDa and is a potent endothelial cell mitogen with a role in proliferation, migration, and tube formation leading to angiogenic growth of new blood vessels. VEGF-A is typically expressed and secreted by a variety of tissues including the retinal pigmented epithelium (RPE), retinal tissues, astrocytes, Müller cells, photoreceptor cells, endothelial cells (e.g., vascular endothelial cells), retinal blood vessels (e.g., including endothelial cells and vascular smooth muscle cells), choroid tissue, e.g., a choroid vessel, and ganglion cells. Several angiogenic ocular disorders are associated with pathological angiogenesis, including wet AMD, DR, DME, RVO, MEfRVO, ROP, and mCNV. Without wishing to be bound by theory, VEGF-A may exacerbate the pathogenesis of angiogenic ocular disorders, e.g., by increasing vascular permeability and promoting neovascularization.

The following description discloses how to make and use compositions containing iRNAs to modulate (e.g., inhibit) the expression of VEGF-A, as well as compositions and methods for treating disorders related to expression of VEGF-A.

In some aspects, pharmaceutical compositions containing VEGF-A iRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of VEGF-A, and methods of using the pharmaceutical compositions to treat disorders related to expression of VEGF-A (e.g., angiogenic ocular disorders) are featured herein.

I. Definitions

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary from, for example, between 1% and 15% of the stated number or numerical range.

The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 17 nucleotides of a 20-nucleotide nucleic acid molecule” means that 17, 18, 19, or 20 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with mismatches to a target site of “no more than 2 nucleotides” has a 2, 1, or 0 mismatches. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.

As used herein, “up to” as in “up to 10” is understood as up to and including 10, i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Ranges provided herein are understood to include all individual integer values and all subranges within the ranges.

The terms “activate,” “enhance,” “up-regulate the expression of,” “increase the expression of,” and the like, in so far as they refer to a VEGF-A gene, herein refer to the at least partial activation of the expression of a VEGF-A gene, as manifested by an increase in the amount of VEGF-A mRNA, which may be isolated from or detected in a first cell or group of cells in which a VEGF-A gene is transcribed and which has or have been treated such that the expression of a VEGF-A gene is increased, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).

In some embodiments, expression of a VEGF-A gene is activated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA as described herein. In some embodiments, a VEGF-A gene is activated by at least about 60%, 70%, or 80% by administration of an iRNA featured in the disclosure. In some embodiments, expression of a VEGF-A gene is activated by at least about 85%, 90%, or 95% or more by administration of an iRNA as described herein. In some embodiments, the VEGF-A gene expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold or more in cells treated with an iRNA as described herein compared to the expression in an untreated cell. Activation of expression by small dsRNAs is described, for example, in Li et al., 2006 Proc. Natl. Acad. Sci. U.S.A. 103:17337-42, and in US2007/0111963 and US2005/226848, each of which is incorporated herein by reference.

The terms “silence,” “inhibit expression of,” “down-regulate expression of,” “suppress expression of,” and the like, in so far as they refer to VEGF-A, herein refer to the at least partial suppression of the expression of VEGF-A, as assessed, e.g., based on VEGF-A mRNA expression, VEGF-A protein expression, or another parameter functionally linked to VEGF-A expression. For example, inhibition of VEGF-A expression may be manifested by a reduction of the amount of VEGF-A mRNA which may be isolated from or detected in a first cell or group of cells in which VEGF-A is transcribed and which has or have been treated such that the expression of VEGF-A is inhibited, as compared to a control. The control may be a second cell or group of cells substantially identical to the first cell or group of cells, except that the second cell or group of cells have not been so treated (control cells). The degree of inhibition is usually expressed as a percentage of a control level, e.g.,

$\frac{\left( {{mRNA}{in}{control}{cells}} \right) - \left( {{mRNA}{in}{treated}{cells}} \right)}{\left( {{mRNA}{in}{control}{cells}} \right)}{\bullet 100}\%$

Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to VEGF-A expression, e.g., the amount of protein encoded by a VEGF-A gene. The reduction of a parameter functionally linked to VEGF-A expression may similarly be expressed as a percentage of a control level. In principle, VEGF-A silencing may be determined in any cell expressing VEGF-A, either constitutively or by genomic engineering, and by any appropriate assay.

For example, in certain instances, expression of VEGF-A is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA disclosed herein. In some embodiments, VEGF-A is suppressed by at least about 60%, 65%, 70%, 75%, or 80% by administration of an iRNA disclosed herein. In some embodiments, VEGF-A is suppressed by at least about 85%, 90%, 95%, 98%, 99%, or more by administration of an iRNA as described herein.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence.

As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. In some embodiments, the region of complementarity comprises 0, 1, or 2 mismatches.

The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.

Complementary sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a VEGF-A protein). For example, a polynucleotide is complementary to at least a part of a VEGF-A mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding VEGF-A. The term “complementarity” refers to the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

As used herein, the term “region of complementarity” refers to the region of one nucleotide sequence agent that is substantially complementary to another sequence, e.g., the region of a sense sequence and corresponding antisense sequence of a dsRNA, or the antisense strand of an iRNA and a target sequence, e.g., a VEGF-A nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the antisense strand of the iRNA. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- or 3′-terminus of the iRNA agent.

“Contacting,” as used herein, includes directly contacting a cell, as well as indirectly contacting a cell. For example, a cell within a subject may be contacted when a composition comprising an iRNA is administered (e.g., intraocularly, topically, or intravenously) to the subject.

“Introducing into a cell,” when referring to an iRNA, means facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; an iRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be by a β-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or known in the art. As used herein, a “disorder related to VEGF-A expression,” a “disease related to VEGF-A expression,” a “pathological process related to VEGF-A expression,” “a VEGF-A-associated disorder,” “a VEGF-A-associated disease,” or the like includes any condition, disorder, or disease in which VEGF-A expression is altered (e.g., decreased or increased relative to a reference level, e.g., a level characteristic of a non-diseased subject). In some embodiments, VEGF-A expression is decreased. In some embodiments, VEGF-A expression is increased. In some embodiments, the decrease or increase in VEGF-A expression is detectable in a tissue sample from the subject (e.g., in an aqueous ocular fluid sample). The decrease or increase may be assessed relative the level observed in the same individual prior to the development of the disorder or relative to other individual(s) who do not have the disorder. The decrease or increase may be limited to a particular organ, tissue, or region of the body (e.g., the eye). VEGF-A-associated disorders include, but are not limited to, angiogenic ocular disorders.

The term “angiogenic ocular disorder,” as used herein, means any disease of the eye that is caused by or associated with the growth or proliferation of blood vessels or by blood vessel leakage. Non-limiting examples of angiogenic ocular disorders that are treatable using methods provided herein include age-related macular degeneration (e.g., wet AMD, exudative AMD, etc.), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO; e.g., macular edema following RVO (MEfRVO)), branch retinal vein occlusion (BRVO), retinopathy of prematurity (ROP), diabetic macular edema (DME), choroidal neovascularization (CNV; e.g., myopic CNV), iris neovascularization, neovascular glaucoma, post-surgical fibrosis in glaucoma, proliferative retinopathy, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreal neovascularization, pannus, pterygium, vascular retinopathy, von Hippel-Lindau disease, histoplasmosis, and diabetic retinopathies.

The term “double-stranded RNA,” “dsRNA,” or “siRNA” as used herein, refers to an iRNA that includes an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having “sense” and “antisense” orientations with respect to a target RNA. The duplex region can be of any length that permits specific degradation of a desired target RNA, e.g., through a RISC pathway, but will typically range from 9 to 36 base pairs in length, e.g., 15-30 base pairs in length. Considering a duplex between 9 and 36 base pairs, the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNAs generated in the cell by processing with Dicer and similar enzymes are generally in the range of 19-22 base pairs in length. One strand of the duplex region of a dsDNA comprises a sequence that is substantially complementary to a region of a target RNA. The two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules. Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop”) between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. In some embodiments, the two strands are connected covalently by means other than a hairpin loop, and the connecting structure is a linker.

In some embodiments, the iRNA agent may be a “single-stranded siRNA” that is introduced into a cell or organism to inhibit a target mRNA. In some embodiments, single-stranded RNAi agents can bind to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are optionally chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein (e.g., sequences provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A or 18B) may be used as a single-stranded siRNA as described herein and optionally as chemically modified, e.g., as described herein, e.g., by the methods described in Lima et al., (2012) Cell 150:883-894.

In some embodiments, an RNA interference agent includes a single stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in some embodiments, the disclosure relates to a single stranded RNA that promotes the formation of a RISC complex to effect silencing of the target gene.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the terms “deoxyribonucleotide,” “ribonucleotide,” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the disclosure by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the disclosure.

As used herein, the term “iRNA,” “RNAi”, “iRNA agent,” or “RNAi agent” or “RNAi molecule” refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript, e.g., via an RNA-induced silencing complex (RISC) pathway. In some embodiments, an iRNA as described herein effects inhibition of VEGF-A expression, e.g., in a cell or mammal. Inhibition of VEGF-A expression may be assessed based on a reduction in the level of VEGF-A mRNA or a reduction in the level of the VEGF-A protein.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound.

The term “lipophile” or “lipophilic moiety” broadly refers to any compound or chemical moiety having an affinity for lipids. One way to characterize the lipophilicity of the lipophilic moiety is by the octanol-water partition coefficient, log K_(ow), where K_(ow) is the ratio of a chemical's concentration in the octanol-phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it may also be predicted by using coefficients attributed to the structural components of a chemical which are calculated using first-principle or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure of the tendency of the substance to prefer a non-aqueous or oily milieu rather than water (i.e. its hydrophilic/lipophilic balance). In principle, a chemical substance is lipophilic in character when its log K_(ow) exceeds 0. Typically, the lipophilic moiety possesses a log K_(ow) exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10. For instance, the log K_(ow) of 6-amino hexanol, for instance, is predicted to be approximately 0.7. Using the same method, the log K_(ow) of cholesteryl N-(hexan-6-ol) carbamate is predicted to be 10.7.

The lipophilicity of a molecule can change with respect to the functional group it carries. For instance, adding a hydroxyl group or amine group to the end of a lipophilic moiety can increase or decrease the partition coefficient (e.g., log K_(ow)) value of the lipophilic moiety.

Alternatively, the hydrophobicity of the double-stranded RNAi agent, conjugated to one or more lipophilic moieties, can be measured by its protein binding characteristics. For instance, in certain embodiments, the unbound fraction in the plasma protein binding assay of the double-stranded RNAi agent could be determined to positively correlate to the relative hydrophobicity of the double-stranded RNAi agent, which could then positively correlate to the silencing activity of the double-stranded RNAi agent.

In some embodiments, the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol of this binding assay is illustrated in detail in, e.g., PCT/US2019/031170. The hydrophobicity of the double-stranded RNAi agent, measured by fraction of unbound siRNA in the binding assay, exceeds 0.15, exceeds 0.2, exceeds 0.25, exceeds 0.3, exceeds 0.35, exceeds 0.4, exceeds 0.45, or exceeds 0.5 for an enhanced in vivo delivery of siRNA.

Accordingly, conjugating the lipophilic moieties to the internal position(s) of the double-stranded RNAi agent provides optimal hydrophobicity for the enhanced in vivo delivery of siRNA.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., a RNAi agent or a plasmid from which a RNAi agent is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

As used herein, the term “modulate the expression of,” refers to an at least partial “inhibition” or partial “activation” of a gene (e.g., VEGF-A gene) expression in a cell treated with an iRNA composition as described herein compared to the expression of the corresponding gene in a control cell. A control cell includes an untreated cell, or a cell treated with a non-targeting control iRNA.

The skilled artisan will recognize that the term “RNA molecule” or “ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a “ribonucleoside” includes a nucleoside base and a ribose sugar, and a “ribonucleotide” is a ribonucleoside with one, two or three phosphate moieties or analogs thereof (e.g., phosphorothioate). However, the terms “ribonucleoside” and “ribonucleotide” can be considered to be equivalent as used herein. The RNA can be modified in the nucleobase structure, in the ribose structure, or in the ribose-phosphate backbone structure, e.g., as described herein below. However, the molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex. As non-limiting examples, an RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2′-O-methyl modified nucleoside, a nucleoside comprising a 5′ phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, an acyclic nucleoside, a glycol nucleotide, a 2′-deoxy-2′-fluoro modified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, or in combination, an RNA molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the dsRNA molecule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule. In some embodiments, modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA, e.g., via a RISC pathway. For clarity, it is understood that the term “iRNA” does not encompass a naturally occurring double stranded DNA molecule or a 100% deoxynucleoside-containing DNA molecule.

In some aspects, a modified ribonucleoside includes a deoxyribonucleoside. In such an instance, an iRNA agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA. In certain embodiments, the RNA molecule comprises a percentage of deoxyribonucleosides of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or higher (but not 100%) deoxyribonucleosides, e.g., in one or both strands.

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′ end, 3′ end or both ends of either an antisense or sense strand of a dsRNA.

In some embodiments, the antisense strand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/or the 5′ end. In some embodiments, the sense strand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/or the 5′ end. In some embodiments, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a therapeutic agent (e.g., an iRNA) and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an agent (e.g., iRNA) effective to produce the intended pharmacological, therapeutic or preventive result. For example, in a method of treating a disorder related to VEGF-A expression (e.g., an angiogenic ocular disorder), an effective amount includes an amount effective to reduce one or more symptoms associated with the disorder (e.g., an amount effective to (a) inhibit angiogenesis; (b) inhibit or reduces the expression or activity of VEGF-A; (c) inhibit choroidal neovascularization; (d) inhibit growth of new blood vessels in the choriocapillaris; (e) reduce retinal thickness; (f) increase visual acuity; or (g) reduce intraocular inflammation) or an amount effective to reduce the risk of developing conditions associated with the disorder. For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to obtain at least a 10% reduction in that parameter. For example, a therapeutically effective amount of an iRNA targeting VEGF-A can reduce a level of VEGF-A mRNA or a level of VEGF-A protein by any measurable amount, e.g., by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Agents included in drug formulations are described further herein below.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 2006/0240093, 2007/0135372, and in International Application No. WO 2009/082817. These applications are incorporated herein by reference in their entirety. In some embodiments, the SNALP is a SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

As used herein, a “subject” to be treated according to the methods described herein, includes a human or non-human animal, e.g., a mammal. The mammal may be, for example, a rodent (e.g., a rat or mouse) or a primate (e.g., a monkey). In some embodiments, the subject is a human.

A “subject in need thereof” includes a subject having, suspected of having, or at risk of developing a disorder related to VEGF-A expression, e.g., overexpression (e.g., an angiogenic ocular disorder). In some embodiments, the subject has, or is suspected of having, a disorder related to VEGF-A expression or overexpression. In some embodiments, the subject is at risk of developing a disorder related to VEGF-A expression or overexpression.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene, e.g., VEGF-A, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween. As non-limiting examples, the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.

As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” and the like refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of any disorder or pathological process related to VEGF-A expression (e.g., an angiogenic ocular disorder). The specific amount that is therapeutically effective may vary depending on factors known in the art, such as, for example, the type of disorder or pathological process, the patient's history and age, the stage of the disorder or pathological process, and the administration of other therapies.

In the context of the present disclosure, the terms “treat,” “treatment,” and the like mean to prevent, delay, relieve or alleviate at least one symptom associated with a disorder related to VEGF-A expression, or to slow or reverse the progression or anticipated progression of such a disorder. For example, the methods featured herein, when employed to treat an angiogenic ocular disorder, may serve to reduce or prevent one or more symptoms of the angiogenic ocular disorder, as described herein, or to reduce the risk or severity of associated conditions. Thus, unless the context clearly indicates otherwise, the terms “treat,” “treatment,” and the like are intended to encompass prophylaxis, e.g., prevention of disorders and/or symptoms of disorders related to VEGF-A expression. Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment.

By “lower” in the context of a disease marker or symptom is meant any decrease, e.g., a statistically or clinically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. The decrease can be down to a level accepted as within the range of normal for an individual without such disorder.

As used herein, “VEGF-A” refers to “vascular endothelial growth factor A” the corresponding mRNA (“VEGF-A mRNA”), or the corresponding protein (“VEGF-A protein”). The sequence of a human VEGF-A mRNA transcript can be found at SEQ ID NO: 1.

II. iRNA Agents

Described herein are iRNA agents that modulate (e.g., inhibit) the expression of VEGF-A.

In some embodiments, the iRNA agent activates the expression of VEGF-A in a cell or mammal.

In some embodiments, the iRNA agent includes double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of VEGF-A in a cell or in a subject (e.g., in a mammal, e.g., in a human), where the dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of VEGF-A, and where the region of complementarity is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and where the dsRNA, upon contact with a cell expressing VEGF-A, inhibits the expression of VEGF-A, e.g., by at least 10%, 20%, 30%, 40%, or 50%.

The modulation (e.g., inhibition) of expression of VEGF-A can be assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by Western blot. Expression of VEGF-A in cell culture, such as in COS cells, ARPE-19 cells, hTERT RPE-1 cells, HeLa cells, primary hepatocytes, HepG2 cells, primary cultured cells or in a biological sample from a subject can be assayed by measuring VEGF-A mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by immunofluorescence analysis, using, for example, Western Blotting or flow cytometric techniques.

A dsRNA typically includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) typically includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of VEGF-A. The other strand (the sense strand) typically includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive.

In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, e.g., 15-30 nucleotides in length.

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of 9 to 36, e.g., 15-30 base pairs. Thus, in some embodiments, to the extent that it becomes processed to a functional duplex of e.g., 15-30 base pairs that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in some embodiments, then, an miRNA is a dsRNA. In some embodiments, a dsRNA is not a naturally occurring miRNA. In some embodiments, an iRNA agent useful to target VEGF-A expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein may further include one or more single-stranded nucleotide overhangs. The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

In some embodiments, VEGF-A is a human VEGF-A.

In specific embodiments, the dsRNA comprises a sense strand that comprises or consists of a sense sequence selected from the sense sequences provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A or 18B and an antisense strand that comprises or consists of an antisense sequence selected from the antisense sequences provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A or 18B.

In some aspects, a dsRNA will include at least sense and antisense nucleotide sequences, whereby the sense strand is selected from the sequences provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A or 18B and the corresponding antisense strand is selected from the sequences provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A or 18B.

In these aspects, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated by the expression of VEGF-A. As such, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand, and the second oligonucleotide is described as the corresponding antisense strand. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

The skilled person is well aware that dsRNAs having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can be effective as well.

In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B, dsRNAs described herein can include at least one strand of a length of minimally 19 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B minus only a few nucleotides on one or both ends will be similarly effective as compared to the dsRNAs described above.

In some embodiments, the dsRNA has a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A or 18B.

In some embodiments, the dsRNA has an antisense sequence that comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides of an antisense sequence provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A or 18B and a sense sequence that comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides of a corresponding sense sequence provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A or 18B.

In some embodiments, the dsRNA comprises an antisense sequence that comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of an antisense sequence provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A or 18B and a sense sequence that comprises at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides of a corresponding sense sequence provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A or 18B.

In some such embodiments, the dsRNA, although it comprises only a portion of the sequences provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A or 18B is equally effective in inhibiting a level of VEGF-A expression as is a dsRNA that comprises the full-length sequences provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A or 18B. In some embodiments, the dsRNA differs in its inhibition of a level of expression of VEGF-A by not more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% inhibition compared with a dsRNA comprising the full sequence disclosed herein.

The iRNAs of Tables 5A and 5B were designed based on rat VEGF-A sequence. Without wishing to be bound by theory, VEGF-A sequence is conserved sufficiently between species such that certain iRNAs designed based on a rodent sequence have activity against a primate VEGF-A. Working Example 2 herein gives evidence of iRNAs designed based on a rodent sequence having activity against cynomolgus monkey VEGF-A.

Consequently, in some embodiments, an iRNA of Table 5A or Table 5B decreases VEGF-A protein or VEGF-A mRNA levels in a cell. In some embodiments, the cell is a rodent cell (e.g., a rat cell), or a primate cell (e.g., a cynomolgus monkey cell or a human cell). In some embodiments, VEGF-A protein or VEGF-F mRNA levels are reduced by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the iRNA of Table 5A or 5B that inhibits VEGF-A in a human cell has less than 5, 4, 3, 2, or 1 mismatches to the corresponding portion of human VEGF-A. In some embodiments, the iRNA of Table 5A or 5B that inhibits VEGF-A in a human cell has no mismatches to the corresponding portion of human VEGF-A.

iRNAs designed based on rodent sequences can have utility, e.g., for inhibiting VEGF-A in human cells, e.g., for therapeutic purposes, or for inhibiting VEGF-A in rodent cells, e.g., for research characterizing VEGF-A in a rodent model.

In some embodiments, an iRNA described herein comprises an antisense strand comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2. In some embodiments, an iRNA described herein comprises a sense strand comprising at least 15 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

A human VEGF-A mRNA may have the sequence of SEQ ID NO: 1 provided herein. Homo sapiens vascular endothelial growth factor A (VEGFA), transcript variant 1, mRNA

(SEQ ID NO: 1) TCGCGGAGGCTTGGGGCAGCCGGGTAGCTCGGAGGTCGTGGCGCTGGGGG CTAGCACCAGCGCTCTGTCGGGAGGCGCAGCGGTTAGGTGGACCGGTCAG CGGACTCACCGGCCAGGGCGCTCGGTGCTGGAATTTGATATTCATTGATC CGGGTTTTATCCCTCTTCTTTTTTCTTAAACATTTTTTTTTAAAACTGTA TTGTTTCTCGTTTTAATTTATTTTTGCTTGCCATTCCCCACTTGAATCGG GCCGACGGCTTGGGGAGATTGCTCTACTTCCCCAAATCACTGTGGATTTT GGAAACCAGCAGAAAGAGGAAAGAGGTAGCAAGAGCTCCAGAGAGAAGTC GAGGAAGAGAGAGACGGGGTCAGAGAGAGCGCGCGGGCGTGCGAGCAGCG AAAGCGACAGGGGCAAAGTGAGTGACCTGCTTTTGGGGGTGACCGCCGGA GCGCGGCGTGAGCCCTCCCCCTTGGGATCCCGCAGCTGACCAGTCGCGCT GACGGACAGACAGACAGACACCGCCCCCAGCCCCAGCTACCACCTCCTCC CCGGCCGGCGGCGGACAGTGGACGCGGCGGCGAGCCGCGGGCAGGGGCCG GAGCCCGCGCCCGGAGGCGGGGTGGAGGGGGTCGGGGCTCGCGGCGTCGC ACTGAAACTTTTCGTCCAACTTCTGGGCTGTTCTCGCTTCGGAGGAGCCG TGGTCCGCGCGGGGGAAGCCGAGCCGAGCGGAGCCGCGAGAAGTGCTAGC TCGGGCCGGGAGGAGCCGCAGCCGGAGGAGGGGGAGGAGGAAGAAGAGAA GGAAGAGGAGAGGGGGCCGCAGTGGCGACTCGGCGCTCGGAAGCCGGGCT CATGGACGGGTGAGGCGGCGGTGTGCGCAGACAGTGCTCCAGCCGCGCGC GCTCCCCAGGCCCTGGCCCGGGCCTCGGGCCGGGGAGGAAGAGTAGCTCG CCGAGGCGCCGAGGAGAGCGGGCCGCCCCACAGCCCGAGCCGGAGAGGGA GCGCGAGCCGCGCCGGCCCCGGTCGGGCCTCCGAAACCATGAACTTTCTG CTGTCTTGGGTGCATTGGAGCCTTGCCTTGCTGCTCTACCTCCACCATGC CAAGTGGTCCCAGGCTGCACCCATGGCAGAAGGAGGAGGGCAGAATCATC ACGAAGTGGTGAAGTTCATGGATGTCTATCAGCGCAGCTACTGCCATCCA ATCGAGACCCTGGTGGACATCTTCCAGGAGTACCCTGATGAGATCGAGTA CATCTTCAAGCCATCCTGTGTGCCCCTGATGCGATGCGGGGGCTGCTGCA ATGACGAGGGCCTGGAGTGTGTGCCCACTGAGGAGTCCAACATCACCATG CAGATTATGCGGATCAAACCTCACCAAGGCCAGCACATAGGAGAGATGAG CTTCCTACAGCACAACAAATGTGAATGCAGACCAAAGAAAGATAGAGCAA GACAAGAAAAAAAATCAGTTCGAGGAAAGGGAAAGGGGCAAAAACGAAAG CGCAAGAAATCCCGGTATAAGTCCTGGAGCGTGTACGTTGGTGCCCGCTG CTGTCTAATGCCCTGGAGCCTCCCTGGCCCCCATCCCTGTGGGCCTTGCT CAGAGCGGAGAAAGCATTTGTTTGTACAAGATCCGCAGACGTGTAAATGT TCCTGCAAAAACACAGACTCGCGTTGCAAGGCGAGGCAGCTTGAGTTAAA CGAACGTACTTGCAGATGTGACAAGCCGAGGCGGTGAGCCGGGCAGGAGG AAGGAGCCTCCCTCAGGGTTTCGGGAACCAGATCTCTCACCAGGAAAGAC TGATACAGAACGATCGATACAGAAACCACGCTGCCGCCACCACACCATCA CCATCGACAGAACAGTCCTTAATCCAGAAACCTGAAATGAAGGAAGAGGA GACTCTGCGCAGAGCACTTTGGGTCCGGAGGGCGAGACTCCGGCGGAAGC ATTCCCGGGCGGGTGACCCAGCACGGTCCCTCTTGGAATTGGATTCGCCA TTTTATTTTTCTTGCTGCTAAATCACCGAGCCCGGAAGATTAGAGAGTTT TATTTCTGGGATTCCTGTAGACACACCCACCCACATACATACATTTATAT ATATATATATTATATATATATAAAAATAAATATCTCTATTTTATATATAT AAAATATATATATTCTTTTTTTAAATTAACAGTGCTAATGTTATTGGTGT CTTCACTGGATGTATTTGACTGCTGTGGACTTGAGTTGGGAGGGGAATGT TCCCACTCAGATCCTGACAGGGAAGAGGAGGAGATGAGAGACTCTGGCAT GATCTTTTTTTTGTCCCACTTGGTGGGGCCAGGGTCCTCTCCCCTGCCCA GGAATGTGGAAGGCCAGGGCATGGGGGCAAATATGACCCAGTTTTGGGAA CACCGACAAACCCAGCCCTGGCGCTGAGCCTCTCTACCCCAGGTCAGACG GACAGAAAGACAGATCACAGGTACAGGGATGAGGACACCGGCTCTGACCA GGAGTTTGGGGAGCTTCAGGACATTGCTGTGCTTTGGGGATTCCCTCCAC ATGCTGCACGCGCATCTCGCCCCCAGGGGCACTGCCTGGAAGATTCAGGA GCCTGGGCGGCCTTCGCTTACTCTCACCTGCTTCTGAGTTGCCCAGGAGA CCACTGGCAGATGTCCCGGCGAAGAGAAGAGACACATTGTTGGAAGAAGC AGCCCATGACAGCTCCCCTTCCTGGGACTCGCCCTCATCCTCTTCCTGCT CCCCTTCCTGGGGTGCAGCCTAAAAGGACCTATGTCCTCACACCATTGAA ACCACTAGTTCTGTCCCCCCAGGAGACCTGGTTGTGTGTGTGTGAGTGGT TGACCTTCCTCCATCCCCTGGTCCTTCCCTTCCCTTCCCGAGGCACAGAG AGACAGGGCAGGATCCACGTGCCCATTGTGGAGGCAGAGAAAAGAGAAAG TGTTTTATATACGGTACTTATTTAATATCCCTTTTTAATTAGAAATTAAA ACAGTTAATTTAATTAAAGAGTAGGGTTTTTTTTCAGTATTCTTGGTTAA TATTTAATTTCAACTATTTATGAGATGTATCTTTTGCTCTCTCTTGCTCT CTTATTTGTACCGGTTTTTGTATATAAAATTCATGTTTCCAATCTCTCTC TCCCTGATCGGTGACAGTCACTAGCTTATCTTGAACAGATATTTAATTTT GCTAACACTCAGCTCTGCCCTCCCCGATCCCCTGGCTCCCCAGCACACAT TCCTTTGAAATAAGGTTTCAATATACATCTACATACTATATATATATTTG GCAACTTGTATTTGTGTGTATATATATATATATATGTTTATGTATATATG TGATTCTGATAAAATAGACATTGCTATTCTGTTTTTTATATGTAAAAACA AAACAAGAAAAAATAGAGAATTCTACATACTAAATCTCTCTCCTTTTTTA ATTTTAATATTTGTTATCATTTATTTATTGGTGCTACTGTTTATCCGTAA TAATTGTGGGGAAAAGATATTAACATCACGTCTTTGTCTCTAGTGCAGTT TTTCGAGATATTCCGTAGTACATATTTATTTTTAAACAACGACAAAGAAA TACAGATATATCTTAAAAAAAAAAAAGCATTTTGTATTAAAGAATTTAAT TCTGATCTCAAAAAAAAAAAAAAAAAA 

The reverse complement of SEQ ID NO: 1 is provided as SEQ ID NO: 2 herein:

(SEQ ID NO: 2) TTTTTTTTTTTTTTTTTTGAGATCAGAATTAAATTCTTTAATACAAAATG CTTTTTTTTTTTTAAGATATATCTGTATTTCTTTGTCGTTGTTTAAAAAT AAATATGTACTACGGAATATCTCGAAAAACTGCACTAGAGACAAAGACGT GATGTTAATATCTTTTCCCCACAATTATTACGGATAAACAGTAGCACCAA TAAATAAATGATAACAAATATTAAAATTAAAAAAGGAGAGAGATTTAGTA TGTAGAATTCTCTATTTTTTCTTGTTTTGTTTTTACATATAAAAAACAGA ATAGCAATGTCTATTTTATCAGAATCACATATATACATAAACATATATAT ATATATATACACACAAATACAAGTTGCCAAATATATATATAGTATGTAGA TGTATATTGAAACCTTATTTCAAAGGAATGTGTGCTGGGGAGCCAGGGGA TCGGGGAGGGCAGAGCTGAGTGTTAGCAAAATTAAATATCTGTTCAAGAT AAGCTAGTGACTGTCACCGATCAGGGAGAGAGAGATTGGAAACATGAATT TTATATACAAAAACCGGTACAAATAAGAGAGCAAGAGAGAGCAAAAGATA CATCTCATAAATAGTTGAAATTAAATATTAACCAAGAATACTGAAAAAAA ACCCTACTCTTTAATTAAATTAACTGTTTTAATTTCTAATTAAAAAGGGA TATTAAATAAGTACCGTATATAAAACACTTTCTCTTTTCTCTGCCTCCAC AATGGGCACGTGGATCCTGCCCTGTCTCTCTGTGCCTCGGGAAGGGAAGG GAAGGACCAGGGGATGGAGGAAGGTCAACCACTCACACACACACAACCAG GTCTCCTGGGGGGACAGAACTAGTGGTTTCAATGGTGTGAGGACATAGGT CCTTTTAGGCTGCACCCCAGGAAGGGGAGCAGGAAGAGGATGAGGGCGAG TCCCAGGAAGGGGAGCTGTCATGGGCTGCTTCTTCCAACAATGTGTCTCT TCTCTTCGCCGGGACATCTGCCAGTGGTCTCCTGGGCAACTCAGAAGCAG GTGAGAGTAAGCGAAGGCCGCCCAGGCTCCTGAATCTTCCAGGCAGTGCC CCTGGGGGCGAGATGCGCGTGCAGCATGTGGAGGGAATCCCCAAAGCACA GCAATGTCCTGAAGCTCCCCAAACTCCTGGTCAGAGCCGGTGTCCTCATC CCTGTACCTGTGATCTGTCTTTCTGTCCGTCTGACCTGGGGTAGAGAGGC TCAGCGCCAGGGCTGGGTTTGTCGGTGTTCCCAAAACTGGGTCATATTTG CCCCCATGCCCTGGCCTTGCACATTCCTGGGCAGGGGAGAGGACCCTGGC CCCACCAAGTGGGACAAAAAAAAGATCATGCCAGAGTCTCTCATCTCCTC CTCTTCCCTGTCAGGATCTGAGTGGGAACATTCCCCTCCCAACTCAAGTC CACAGCAGTCAAATACATCCAGTGAAGACACCAATAACATTAGCACTGTT AATTTAAAAAAAGAATATATATATTTTATATATATAAAATAGAGATATTT ATTTTTATATATATATAATATATATATATATAAATGTATGTATGTGGGTG GGTGTGTCTACAGGAATCCCAGAAATAAAACTCTCTAATCTTCCGGGCTC GGTGATTTAGCAGCAAGAAAAATAAAATGGCGAATCCAATTCCAAGAGGG ACCGTGCTGGGTCACCCGCCCGGGAATGCTTCCGCCGGAGTCTCGCCCTC CGGACCCAAAGTGCTCTGCGCAGAGTCTCCTCTTCCTTCATTTCAGGTTT CTGGATTAAGGACTGTTCTGTCGATGGTGATGGTGTGGTGGCGGCAGCGT GGTTTCTGTATCGATCGTTCTGTATCAGTCTTTCCTGGTGAGAGATCTGG TTCCCGAAACCCTGAGGGAGGCTCCTTCCTCCTGCCCGGCTCACCGCCTC GGCTTGTCACATCTGCAAGTACGTTCGTTTAACTCAAGCTGCCTCGCCTT GCAACGCGAGTCTGTGTTTTTGCAGGAACATTTACACGTCTGCGGATCTT GTACAAACAAATGCTTTCTCCGCTCTGAGCAAGGCCCACAGGGATGGGGG CCAGGGAGGCTCCAGGGCATTAGACAGCAGCGGGCACCAACGTACACGCT CCAGGACTTATACCGGGATTTCTTGCGCTTTCGTTTTTGCCCCTTTCCCT TTCCTCGAACTGATTTTTTTTCTTGTCTTGCTCTATCTTTCTTTGGTCTG CATTCACATTTGTTGTGCTGTAGGAAGCTCATCTCTCCTATGTGCTGGCC TTGGTGAGGTTTGATCCGCATAATCTGCATGGTGATGTTGGACTCCTCAG TGGGCACACACTCCAGGCCCTCGTCATTGCAGCAGCCCCCGCATCGCATC AGGGGCACACAGGATGGCTTGAAGATGTACTCGATCTCATCAGGGTACTC CTGGAAGATGTCCACCAGGGTCTCGATTGGATGGCAGTAGCTGCGCTGAT AGACATCCATGAACTTCACCACTTCGTGATGATTCTGCCCTCCTCCTTCT GCCATGGGTGCAGCCTGGGACCACTTGGCATGGTGGAGGTAGAGCAGCAA GGCAAGGCTCCAATGCACCCAAGACAGCAGAAAGTTCATGGTTTCGGAGG CCCGACCGGGGCCGGCGCGGCTCGCGCTCCCTCTCCGGCTCGGGCTGTGG GGCGGCCCGCTCTCCTCGGCGCCTCGGCGAGCTACTCTTCCTCCCCGGCC CGAGGCCCGGGCCAGGGCCTGGGGAGCGCGCGCGGCTGGAGCACTGTCTG CGCACACCGCCGCCTCACCCGTCCATGAGCCCGGCTTCCGAGCGCCGAGT CGCCACTGCGGCCCCCTCTCCTCTTCCTTCTCTTCTTCCTCCTCCCCCTC CTCCGGCTGCGGCTCCTCCCGGCCCGAGCTAGCACTTCTCGCGGCTCCGC TCGGCTCGGCTTCCCCCGCGCGGACCACGGCTCCTCCGAAGCGAGAACAG CCCAGAAGTTGGACGAAAAGTTTCAGTGCGACGCCGCGAGCCCCGACCCC CTCCACCCCGCCTCCGGGCGCGGGCTCCGGCCCCTGCCCGCGGCTCGCCG CCGCGTCCACTGTCCGCCGCCGGCCGGGGAGGAGGTGGTAGCTGGGGCTG GGGGCGGTGTCTGTCTGTCTGTCCGTCAGCGCGACTGGTCAGCTGCGGGA TCCCAAGGGGGAGGGCTCACGCCGCGCTCCGGCGGTCACCCCCAAAAGCA GGTCACTCACTTTGCCCCTGTCGCTTTCGCTGCTCGCACGCCCGCGCGCT CTCTCTGACCCCGTCTCTCTCTTCCTCGACTTCTCTCTGGAGCTCTTGCT ACCTCTTTCCTCTTTCTGCTGGTTTCCAAAATCCACAGTGATTTGGGGAA GTAGAGCAATCTCCCCAAGCCGTCGGCCCGATTCAAGTGGGGAATGGCAA GCAAAAATAAATTAAAACGAGAAACAATACAGTTTTAAAAAAAAATGTTT AAGAAAAAAGAAGAGGGATAAAACCCGGATCAATGAATATCAAATTCCAG CACCGAGCGCCCTGGCCGGTGAGTCCGCTGACCGGTCCACCTAACCGCTG CGCCTCCCGACAGAGCGCTGGTGCTAGCCCCCAGCGCCACGACCTCCGAG CTACCCGGCTGCCCCAAGCCTCCGCGA 

In some embodiments, an iRNA described herein includes at least 15 contiguous nucleotides from one of the sequences provided in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B, and may optionally be coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in VEGF-A.

While a target sequence is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that may serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays described herein or known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., in Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B, further optimization can be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those and sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.

In some embodiments, the disclosure provides an iRNA of any of Tables 2B, 3B, 4B, 5B, 8B, 10B, 14, or 18B that un-modified or un-conjugated. In some embodiments, an RNAi agent of the disclosure has a nucleotide sequence as provided in any of Tables 2A, 3A, 4A, 5A, 8A, 10A, 12, 13 14, and 18A but lacks one or more ligand or moiety shown in the table. A ligand or moiety (e.g., a lipophilic ligand or moiety) can be included in any of the positions provided in the instant application.

An iRNA as described herein can contain one or more mismatches to the target sequence. In some embodiments, an iRNA as described herein contains no more than 3 mismatches. In some embodiments, when the antisense strand of the iRNA contains mismatches to a target sequence, the area of mismatch is not located in the center of the region of complementarity. In some embodiments, when the antisense strand of the iRNA contains mismatches to the target sequence, the mismatch is restricted to be within the last 5 nucleotides from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide iRNA agent RNA strand which is complementary to a region of VEGF-A, the RNA strand generally does not contain any mismatch within the central 13 nucleotides. The methods described herein, or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of VEGF-A. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of VEGF-A is important, especially if the particular region of complementarity in a VEGF-A gene is known to have polymorphic sequence variation within the population.

In some embodiments, at least one end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. In some embodiments, dsRNAs having at least one nucleotide overhang have superior inhibitory properties relative to their blunt-ended counterparts. In some embodiments, the RNA of an iRNA (e.g., a dsRNA) is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the disclosure may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position, or having an acyclic sugar) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in this disclosure include, but are not limited to, RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and RE39464, each of which is herein incorporated by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.

In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the disclosure include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂.

In other embodiments, an iRNA agent comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides (or nucleosides). In certain embodiments, the sense strand or the antisense strand, or both sense strand and antisense strand, include less than five acyclic nucleotides per strand (e.g., four, three, two or one acyclic nucleotides per strand). The one or more acyclic nucleotides can be found, for example, in the double-stranded region, of the sense or antisense strand, or both strands; at the 5′-end, the 3′-end, both of the 5′ and 3′-ends of the sense or antisense strand, or both strands, of the iRNA agent. In some embodiments, one or more acyclic nucleotides are present at positions 1 to 8 of the sense or antisense strand, or both. In some embodiments, one or more acyclic nucleotides are found in the antisense strand at positions 4 to 10 (e.g., positions 6-8) from the 5′-end of the antisense strand. In some embodiments, the one or more acyclic nucleotides are found at one or both 3′-terminal overhangs of the iRNA agent.

The term “acyclic nucleotide” or “acyclic nucleoside” as used herein refers to any nucleotide or nucleoside having an acyclic sugar, e.g., an acyclic ribose. An exemplary acyclic nucleotide or nucleoside can include a nucleobase, e.g., a naturally occurring or a modified nucleobase (e.g., a nucleobase as described herein). In certain embodiments, a bond between any of the ribose carbons (C1, C2, C3, C4, or C5), is independently or in combination absent from the nucleotide. In some embodiments, the bond between C2-C3 carbons of the ribose ring is absent, e.g., an acyclic 2′-3′-seco-nucleotide monomer. In other embodiments, the bond between C1-C2, C3-C4, or C4-05 is absent (e.g., a 1′-2′, 3′-4′ or 4′-5′-seco nucleotide monomer). Exemplary acyclic nucleotides are disclosed in U.S. Pat. No. 8,314,227, incorporated herein by reference in its entirely. For example, an acyclic nucleotide can include any of monomers D-J in FIGS. 1-2 of U.S. Pat. No. 8,314,227. In some embodiments, the acyclic nucleotide includes the following monomer:

wherein Base is a nucleobase, e.g., a naturally occurring or a modified nucleobase (e.g., a nucleobase as described herein).

In certain embodiments, the acyclic nucleotide can be modified or derivatized, e.g., by coupling the acyclic nucleotide to another moiety, e.g., a ligand (e.g., a GalNAc, a cholesterol ligand), an alkyl, a polyamine, a sugar, a polypeptide, among others.

In other embodiments, the iRNA agent includes one or more acyclic nucleotides and one or more LNAs (e.g., an LNA as described herein). For example, one or more acyclic nucleotides and/or one or more LNAs can be present in the sense strand, the antisense strand, or both. The number of acyclic nucleotides in one strand can be the same or different from the number of LNAs in the opposing strand. In certain embodiments, the sense strand and/or the antisense strand comprises less than five LNAs (e.g., four, three, two or one LNAs) located in the double stranded region or a 3′-overhang. In other embodiments, one or two LNAs are located in the double stranded region or the 3′-overhang of the sense strand. Alternatively, or in combination, the sense strand and/or antisense strand comprises less than five acyclic nucleotides (e.g., four, three, two or one acyclic nucleotides) in the double-stranded region or a 3′-overhang. In some embodiments, the sense strand of the iRNA agent comprises one or two LNAs in the 3′-overhang of the sense strand, and one or two acyclic nucleotides in the double-stranded region of the antisense strand (e.g., at positions 4 to 10 (e.g., positions 6-8) from the 5′-end of the antisense strand) of the iRNA agent.

In other embodiments, inclusion of one or more acyclic nucleotides (alone or in addition to one or more LNAs) in the iRNA agent results in one or more (or all) of: (i) a reduction in an off-target effect; (ii) a reduction in passenger strand participation in RNAi; (iii) an increase in specificity of the guide strand for its target mRNA; (iv) a reduction in a microRNA off-target effect; (v) an increase in stability; or (vi) an increase in resistance to degradation, of the iRNA molecule.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-5 aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

An iRNA may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine.

Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.

The RNA of an iRNA can also be modified to include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) bicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the disclosure may include one or more locked nucleic acids (LNAs) (also referred to herein as “locked nucleotides”). In some embodiments, a locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting, e.g., the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, increase thermal stability, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

Examples of bicyclic nucleosides for use in the polynucleotides of the disclosure include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms.

In certain embodiments, the antisense polynucleotide agents of the disclosure include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′; 4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′ (see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The contents of each of the foregoing are incorporated herein by reference for the methods provided therein. Representative U.S. Patents that teach the preparation of locked nucleic acids include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; 7,399,845, and 8,314,227, each of which is herein incorporated by reference in its entirety. Exemplary LNAs include but are not limited to, a 2′, 4′-C methylene bicyclo nucleotide (see for example Wengel et al., International PCT 5 Publication No. WO 00/66604 and WO 99/14226).

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

A RNAi agent of the disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)-O-2′ bridge. In some embodiments, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”

A RNAi agent of the disclosure may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US 2013/0190383; and WO 2013/036868, the contents of each of which are hereby incorporated herein by reference for the methods provided therein. In some embodiments, a RNAi agent of the disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039).

Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the contents of each of which are hereby incorporated herein by reference for the methods provided therein.

In other embodiments, the iRNA agents include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in the iRNA molecules can result in enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands.

Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

Other modifications of a RNAi agent of the disclosure include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of a RNAi agent. Suitable phosphate mimics are disclosed in, for example US 2012/0157511, the contents of which are incorporated herein by reference for the methods provided therein.

iRNA Motifs

In certain aspects of the disclosure, the double-stranded RNAi agents of the disclosure include agents with chemical modifications as disclosed, for example, in WO 2013/075035, the contents of which are incorporated herein by reference for the methods provided therein. As shown herein and in WO 2013/075035, a superior result may be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense strand or antisense strand of an RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the RNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense or antisense strand. The RNAi agent may be optionally conjugated with a lipophilic moiety or ligand, e.g., a C16 moiety or ligand, for instance on the sense strand. The RNAi agent may be optionally modified with a (S)-glycol nucleic acid (GNA) modification, for instance on one or more residues of the antisense strand. The resulting RNAi agents present superior gene silencing activity.

In some embodiments, the sense strand sequence may be represented by formula (I):

5′n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n_(q)3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein N_(b) and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. In some embodiments, YYY is all 2′-F modified nucleotides.

In some embodiments, the N_(a) and/or N_(b) comprise modifications of alternating pattern.

In some embodiments, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11,12 or 11, 12, 13) of the sense strand, the count starting from the 1^(st) nucleotide, from the 5′-end; or optionally, the count starting at the 1_(st) paired nucleotide within the duplex region, from the 5′-end.

In some embodiments, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:

5′n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′  (Ib);

5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q)3′  (Ic); or

5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′  (Id).

When the sense strand is represented by formula (Ib), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Id), each N_(b) independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. In some embodiments, N_(b) 1S 0, 1, 2, 3, 4, 5 or 6. Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula:

5′n_(p)-N_(a)-YYY-N_(a)-n_(q)3′  (Ia).

When the sense strand is represented by formula (Ia), each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

In some embodiments, the antisense strand sequence of the RNAi may be represented by formula (II):

5′n_(q)′-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)_(l)-N′_(a)-n_(p)′3′  (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification;

and

X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one of three identical modification on three consecutive nucleotides.

In some embodiments, the N_(a)′ and/or N_(b)′ comprise modification of alternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the 1^(st) nucleotide, from the 5′-end; or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end. In some embodiments, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In some embodiments, Y′Y′Y′ motif is all 2′-Ome modified nucleotides.

In on embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both 5 k and l are 1.

The antisense strand can therefore be represented by the following formulas:

5′n_(q)′-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n_(p)′3′  (IIb);

5′n_(q)′-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n_(p)′3′  (IIc); or

5′n_(q)′-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N_(a)′-n_(p)′3′  (IId).

When the antisense strand is represented by formula (IIb), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. In some embodiments, N_(b) is 0, 1, 2, 3, 4, 5 or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand may be represented by the formula:

5′n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′-n_(q)′3′  (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, HNA, CeNA, GNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.

In some embodiments, the sense strand of the RNAi agent may contain YYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1^(st) nucleotide from the 5′-end, or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.

In some embodiments the antisense strand may Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1^(st) nucleotide from the 5′-end, or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with an antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.

Accordingly, certain RNAi agents for use in the methods of the disclosure may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex represented by formula (III):

sense: 5′n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n_(q)3′

antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′   (III)

wherein,

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may not be present independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modification on three consecutive nucleotides.

In some embodiments, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In some embodiments, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forming a RNAi duplex include the formulas below:

5′n_(p)-N_(a)-YYY-N_(a)-n_(q)3′

3′n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′n_(q)′5′   (IIIa)

5′n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′

3′n_(p)-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′-n_(q)′5′   (IIIb)

5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q)3′

3′n_(p)-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n_(q)′5′   (IIIc)

5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′

3′n_(p)-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′-n_(q)′5′   (IIId)

When the RNAi agent is represented by formula (IIIa), each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N_(b) independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a), N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b) and N_(b)′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may form a base pair with one of the Y′ nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z′ nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X′ nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X′ nucleotides.

In some embodiments, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, and/or the modification on the X nucleotide is different than the modification on the X′ nucleotide.

In some embodiments, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. In some embodiments, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications and n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In some embodiments, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties, or one or more GalNAc moieties) attached through a bivalent or trivalent branched linker. In some embodiments, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties, or one or more GalNAc moieties) attached through a bivalent or trivalent branched linker.

In some embodiments, when the RNAi agent is represented by formula (IIIa), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties, or one or more GalNAc moieties) attached through a bivalent or trivalent branched linker.

In some embodiments, the RNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In some embodiments, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In some embodiments, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, and one or both of the 3′ ends and are optionally conjugated to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used in the methods of the disclosure. Such publications include WO2007/091269, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520; and U.S. Pat. No. 7,858,769, the contents of each of which are hereby incorporated herein by reference for the methods provided therein. In certain embodiments, the RNAi agents of the disclosure may include GalNAc ligands.

As described in more detail below, the RNAi agent that contains conjugations of one or more carbohydrate moieties to a RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methods of the disclosure is an agent selected from the group of agents listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B. These agents may further comprise a ligand. The ligand can be attached to the sense strand, antisense strand or both strands, at the 3′-end, 5′-end, or both ends. For instance, the ligand may be conjugated to the sense strand, in particular, the 3′-end of the sense strand.

iRNA Conjugates

The iRNA agents disclosed herein can be in the form of conjugates. The conjugate may be attached at any suitable location in the iRNA molecule, e.g., at the 3′ end or the 5′ end of the sense or the antisense strand. The conjugates are optionally attached via a linker.

In some embodiments, an iRNA agent described herein is chemically linked to one or more ligands, moieties or conjugates, which may confer functionality, e.g., by affecting (e.g., enhancing) the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In some embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In some embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Typical ligands will not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazine. Examples of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an a helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as an ocular cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present disclosure as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the disclosure may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present disclosure may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present disclosure, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present disclosure are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipophilic Moieties

In certain embodiments, the lipophilic moiety is an aliphatic, cyclic such as alicyclic, or polycyclic such as polyalicyclic compound, such as a steroid (e.g., sterol) or a linear or branched aliphatic hydrocarbon. The lipophilic moiety may generally comprise a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may comprise various substituents or one or more heteroatoms, such as an oxygen or nitrogen atom. Such lipophilic aliphatic moieties include, without limitation, saturated or unsaturated C₄-C₃₀ hydrocarbon (e.g., C₆-C₁₈ hydrocarbon), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C₁₀ terpenes, C₁₅ sesquiterpenes, C₂₀ diterpenes, C₃₀ triterpenes, and C₄₀ tetraterpenes), and other polyalicyclic hydrocarbons. For instance, the lipophilic moiety may contain a C₄-C₃₀ hydrocarbon chain (e.g., C₄-C₃₀ alkyl or alkenyl). In some embodiments the lipophilic moiety contains a saturated or unsaturated C₆-C₁₈ hydrocarbon chain (e.g., a linear C₆-C₁₈ alkyl or alkenyl). In some embodiments, the lipophilic moiety contains a saturated or unsaturated C₁₆ hydrocarbon chain (e.g., a linear C₁₆ alkyl or alkenyl).

The lipophilic moiety may be attached to the RNAi agent by any method known in the art, including via a functional grouping already present in the lipophilic moiety or introduced into the RNAi agent, such as a hydroxy group (e.g., —CO—CH₂—OH). The functional groups already present in the lipophilic moiety or introduced into the RNAi agent include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

Conjugation of the RNAi agent and the lipophilic moiety may occur, for example, through formation of an ether or a carboxylic or carbamoyl ester linkage between the hydroxy and an alkyl group R—, an alkanoyl group RCO— or a substituted carbamoyl group RNHCO—. The alkyl group R may be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chained or branched; and saturated or unsaturated). Alkyl group R may be a butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl or octadecyl group, or the like.

In some embodiments, the lipophilic moiety is conjugated to the double-stranded RNAi agent via a linker a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.

In another embodiment, the lipophilic moiety is a steroid, such as sterol. Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentanophenanthrene ring system. Steroids include, without limitation, bile acids (e.g., cholic acid, deoxycholic acid and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cationic steroids, such as cortisone. A “cholesterol derivative” refers to a compound derived from cholesterol, for example by substitution, addition or removal of substituents.

In another embodiment, the lipophilic moiety is an aromatic moiety. In this context, the term “aromatic” refers broadly to mono- and polyaromatic hydrocarbons. Aromatic groups include, without limitation, C6-C14 aryl moieties comprising one to three aromatic rings, which may be optionally substituted; “aralkyl” or “arylalkyl” groups comprising an aryl group covalently linked to an alkyl group, either of which may independently be optionally substituted or unsubstituted; and “heteroaryl” groups. As used herein, the term “heteroaryl” refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14n electrons shared in a cyclic array, and having, in addition to carbon atoms, one to about three heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).

As employed herein, a “substituted” alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group is one having one to about four, preferably one to about three, more preferably one or two, non-hydrogen substituents. Suitable substituents include, without limitation, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups.

In some embodiments, the lipophilic moiety is an aralkyl group, e.g., a 2-arylpropanoyl moiety. The structural features of the aralkyl group are selected so that the lipophilic moiety will bind to at least one protein in vivo. In certain embodiments, the structural features of the aralkyl group are selected so that the lipophilic moiety binds to serum, vascular, or cellular proteins. In certain embodiments, the structural features of the aralkyl group promote binding to albumin, an immunoglobulin, a lipoprotein, α-2-macroglubulin, or α-1-glycoprotein.

In certain embodiments, the ligand is naproxen or a structural derivative of naproxen. Procedures for the synthesis of naproxen can be found in U.S. Pat. Nos. 3,904,682 and 4,009,197, which are hereby incorporated by reference in their entirety. Naproxen has the chemical name (S)-6-Methoxy-α-methyl-2-naphthaleneacetic acid and the structure is

In certain embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. Procedures for the synthesis of ibuprofen can be found in U.S. Pat. No. 3,228,831, which is incorporated herein by reference for the methods provided therein. The structure of ibuprofen is

Additional exemplary aralkyl groups are illustrated in U.S. Pat. No. 7,626,014, which is incorporated herein by reference for the methods provided therein.

In another embodiment, suitable lipophilic moieties include lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

In certain embodiments, more than one lipophilic moiety can be incorporated into the double-strand RNAi agent, particularly when the lipophilic moiety has a low lipophilicity or hydrophobicity. In some embodiments, two or more lipophilic moieties are incorporated into the same strand of the double-strand RNAi agent. In some embodiments, each strand of the double-strand RNAi agent has one or more lipophilic moieties incorporated. In some embodiments, two or more lipophilic moieties are incorporated into the same position (i.e., the same nucleobase, same sugar moiety, or same internucleosidic linkage) of the double-strand RNAi agent. This can be achieved by, e.g., conjugating the two or more lipophilic moieties via a carrier, or conjugating the two or more lipophilic moieties via a branched linker, or conjugating the two or more lipophilic moieties via one or more linkers, with one or more linkers linking the lipophilic moieties consecutively.

The lipophilic moiety may be conjugated to the RNAi agent via a direct attachment to the ribosugar of the RNAi agent. Alternatively, the lipophilic moiety may be conjugated to the double-strand RNAi agent via a linker or a carrier.

In certain embodiments, the lipophilic moiety may be conjugated to the RNAi agent via one or more linkers (tethers).

In some embodiments, the lipophilic moiety is conjugated to the double-stranded RNAi agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.

B. Lipid Conjugates

In some embodiments, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can typically bind a serum protein, such as human serum albumin (HSA). An HSA binding ligand allows for vascular distribution of the conjugate to a target tissue. For example, the target tissue can be the eye. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid-based ligand can be used to modulate, e.g., control (e.g., inhibit) the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In some embodiments, the lipid-based ligand binds HSA. For example, the ligand can bind HSA with a sufficient affinity such that distribution of the conjugate to a non-kidney tissue is enhanced. However, the affinity is typically not so strong that the HSA-ligand binding cannot be reversed.

In some embodiments, the lipid-based ligand binds HSA weakly or not at all, such that distribution of the conjugate to the kidney is enhanced. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid-based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, such as a helical cell-permeation agent. In some embodiments, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is typically an α-helical agent, and can have a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 4158). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 4159)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 4160)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 4161)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Typically, the peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the disclosure may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidomimetics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. In some embodiments, conjugates of this ligand target PECAM-1 or VEGF.

An RGD peptide moiety can be used to target a particular cell type, e.g., a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an dsRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver a iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

Carbohydrate Conjugates and Ligands

In some embodiments of the compositions and methods of the disclosure, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In certain embodiments, the compositions and methods of the disclosure include a C16 ligand. In exemplary embodiments, the C16 ligand of the disclosure has the following structure (exemplified here below for a uracil base, yet attachment of the C16 ligand is contemplated for a nucleotide presenting any base (C, G, A, etc.) or possessing any other modification as presented herein, provided that 2′ ribo attachment is preserved) and is attached at the 2′ position of the ribo within a residue that is so modified:

As shown above, a C16 ligand-modified residue presents a straight chain alkyl at the 2′-ribo position of an exemplary residue (here, a Uracil) that is so modified.

In some embodiments, a carbohydrate conjugate of a RNAi agent of the instant disclosure further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell permeation peptide.

Additional carbohydrate conjugates (and linkers) suitable for use in the present disclosure include those described in WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

In certain embodiments, the compositions and methods of the disclosure include a vinyl phosponate (VP) modification of an RNAi agent as described herein. In exemplary embodiments, a vinyl phosphonate of the disclosure has the following structure:

A vinyl phosponate of the instant disclosure may be attached to either the antisense or the sense strand of a dsRNA of the disclosure. In certain preferred embodiments, a vinyl phosphonate of the instant disclosure is attached to the antisense strand of a dsRNA, optionally at the 5′ end of the antisense strand of the dsRNA.

Vinyl phosphate modifications are also contemplated for the compositions and methods of the instant disclosure. An exemplary vinyl phosphate structure is:

In some embodiments, a carbohydrate conjugate comprises a monosaccharide. In some embodiments, the monosaccharide is an N-acetylgalactosamine (GalNAc). GalNAc conjugates, which comprise one or more N-acetylgalactosamine (GalNAc) derivatives, are described, for example, in U.S. Pat. No. 8,106,022, the entire content of which is hereby incorporated herein by reference. In some embodiments, the GalNAc conjugate serves as a ligand that targets the iRNA to particular cells. In some embodiments, the GalNAc conjugate targets the iRNA to liver cells, e.g., by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be attached via a linker, e.g., a bivalent or trivalent branched linker. In some embodiments the GalNAc conjugate is conjugated to the 3′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3′ end of the sense strand) via a linker, e.g., a linker as described herein.

In some embodiments, the GalNAc conjugate is

In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic, wherein X is O or S:

In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below:

In some embodiments, a carbohydrate conjugate for use in the compositions and methods of the disclosure is selected from the group consisting of:

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.

In some embodiments, an iRNA of the disclosure is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the disclosure include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

E. Thermally Destabilizing Modifications

In certain embodiments, a dsRNA molecule can be optimized for RNA interference by incorporating thermally destabilizing modifications in the seed region of the antisense strand (i.e., at positions 2-9 of the 5′-end of the antisense strand) to reduce or inhibit off-target gene silencing. It has been discovered that dsRNAs with an antisense strand comprising at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5′ end, of the antisense strand have reduced off-target gene silencing activity. Accordingly, in some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five, or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region of the antisense strand. In some embodiments, one or more thermally destabilizing modification(s) of the duplex is/are located in positions 2-9, or preferably positions 4-8, from the 5′-end of the antisense strand. In some further embodiments, the thermally destabilizing modification(s) of the duplex is/are located at position 6, 7, or 8 from the 5′-end of the antisense strand. In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5′-end of the antisense strand. The term “thermally destabilizing modification(s)” includes modification(s) that would result with a dsRNA with a lower overall melting temperature (Tm) (preferably a Tm with one, two, three, or four degrees lower than the Tm of the dsRNA without having such modification(s). In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5, or 9 from the 5′-end of the antisense strand.

The thermally destabilizing modifications can include, but are not limited to, abasic modification; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2′-deoxy modification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA).

Exemplified abasic modifications include, but are not limited to, the following:

Wherein R═H, Me, Et or OMe; R′═H, Me, Et or OMe; R″═H, Me, Et or OMe

wherein B is a modified or unmodified nucleobase.

Exemplified sugar modifications include, but are not limited to the following:

wherein B is a modified or unmodified nucleobase.

In some embodiments the thermally destabilizing modification of the duplex is selected from the group consisting of:

wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic.

The term “acyclic nucleotide” refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., C1′-C2′, C2′-C3′, C3′-C4′, C4′-O4′, or C1′-O4′) is absent or at least one of ribose carbons or oxygen (e.g., C1′, C2′, C3′, C4′, or O4′) are independently or in combination absent from the nucleotide. In some embodiments, acyclic nucleotide is

wherein B is a modified or unmodified nucleobase, R¹ and R² independently are H, halogen, OR₃, or alkyl; and R₃ is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term “UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomers with bonds between C1′-C4′ being removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar is removed (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are hereby incorporated by reference in their entirety). The acyclic derivative provides greater backbone flexibility without affecting the Watson-Crick pairings. The acyclic nucleotide can be linked via 2′-5′ or 3′-5′ linkage.

The term ‘GNA’ refers to glycol nucleic acid which is a polymer similar to DNA or RNA but differing in the composition of its “backbone” in that is composed of repeating glycerol units linked by phosphodiester bonds:

The thermally destabilizing modification of the duplex can be mismatches (i.e., noncomplementary base pairs) between the thermally destabilizing nucleotide and the opposing nucleotide in the opposite strand within the dsRNA duplex. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Other mismatch base pairings known in the art are also amenable to the present invention. A mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatch base pairing can occur between the nucleobases from respective nucleotides independent of the modifications on the ribose sugars of the nucleotides. In certain embodiments, the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2′-deoxy nucleobase; e.g., the 2′-deoxy nucleobase is in the sense strand.

In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes nucleotides with impaired W-C H-bonding to complementary base on the target mRNA, such as:

More examples of abasic nucleotide, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications have been described in detail in WO 2011/133876, which is herein incorporated by reference in its entirety.

The thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications.

In some embodiments, the thermally destabilizing modification of the duplex includes nucleotides with non-canonical bases such as, but not limited to, nucleobase modifications with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand. These nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010/0011895, which is herein incorporated by reference in its entirety. Exemplary nucleobase modifications are:

In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes one or more α-nucleotide complementary to the base on the target mRNA, such as:

wherein R is H, OH, OCH₃, F, NH₂, NHMe, NMe₂ or O-alkyl.

Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages are:

The alkyl for the R group can be a C₁-C₆alkyl. Specific alkyls for the R group include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.

As the skilled artisan will recognize, in view of the functional role of nucleobases is defining specificity of a RNAi agent of the disclosure, while nucleobase modifications can be performed in the various manners as described herein, e.g., to introduce destabilizing modifications into a RNAi agent of the disclosure, e.g., for purpose of enhancing on-target effect relative to off-target effect, the range of modifications available and, in general, present upon RNAi agents of the disclosure tends to be much greater for non-nucleobase modifications, e.g., modifications to sugar groups or phosphate backbones of polyribonucleotides. Such modifications are described in greater detail in other sections of the instant disclosure and are expressly contemplated for RNAi agents of the disclosure, either possessing native nucleobases or modified nucleobases as described above or elsewhere herein.

In addition to the antisense strand comprising a thermally destabilizing modification, the dsRNA can also comprise one or more stabilizing modifications. For example, the dsRNA can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitations, the stabilizing modifications all can be present in one strand. In some embodiments, both the sense and the antisense strands comprise at least two stabilizing modifications. The stabilizing modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the stabilizing modification can occur on every nucleotide on the sense strand or antisense strand; each stabilizing modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both stabilizing modification in an alternating pattern. The alternating pattern of the stabilizing modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the stabilizing modifications on the sense strand can have a shift relative to the alternating pattern of the stabilizing modifications on the antisense strand.

In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitations, a stabilizing modification in the antisense strand can be present at any positions.

In some embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5′-end. In some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5′-end. In still some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 14, and 16 from the 5′-end.

In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification. For example, the stabilizing modification can be the nucleotide at the 5′-end or the 3′-end of the destabilizing modification, i.e., at position −1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5′-end and the 3′-end of the destabilizing modification, i.e., positions −1 and +1 from the position of the destabilizing modification.

In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3′-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.

In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the sense strand can be present at any positions. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5′-end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from the 5′-end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three, or four stabilizing modifications.

In some embodiments, the sense strand does not comprise a stabilizing modification in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.

Exemplary thermally stabilizing modifications include, but are not limited to, 2′-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

In some embodiments, the dsRNA of the disclosure comprises at least four (e.g., four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, the 2′-fluoro nucleotides all can be present in one strand. In some embodiments, both the sense and the antisense strands comprise at least two 2′-fluoro nucleotides. The 2′-fluoro modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the 2′-fluoro modification can occur on every nucleotide on the sense strand or antisense strand; each 2′-fluoro modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both 2′-fluoro modifications in an alternating pattern. The alternating pattern of the 2′-fluoro modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the 2′-fluoro modifications on the sense strand can have a shift relative to the alternating pattern of the 2′-fluoro modifications on the antisense strand.

In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, a 2′-fluoro modification in the antisense strand can be present at any positions. In some embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5′-end. In some other embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5′-end. In still some other embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 14, and 16 from the 5′-end.

In some embodiments, the antisense strand comprises at least one 2′-fluoro nucleotide adjacent to the destabilizing modification. For example, the 2′-fluoro nucleotide can be the nucleotide at the 5′-end or the 3′-end of the destabilizing modification, i.e., at position −1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a 2′-fluoro nucleotide at each of the 5′-end and the 3′-end of the destabilizing modification, i.e., positions −1 and +1 from the position of the destabilizing modification.

In some embodiments, the antisense strand comprises at least two 2′-fluoro nucleotides at the 3′-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.

In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, a 2′-fluoro modification in the sense strand can be present at any positions. In some embodiments, the antisense comprises 2′-fluoro nucleotides at positions 7, 10, and 11 from the 5′-end. In some other embodiments, the sense strand comprises 2′-fluoro nucleotides at positions 7, 9, 10, and 11 from the 5′-end. In some embodiments, the sense strand comprises 2′-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some other embodiments, the sense strand comprises 2′-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three, or four 2′-fluoro nucleotides.

In some embodiments, the sense strand does not comprise a 2′-fluoro nucleotide in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.

In some embodiments, the dsRNA molecule of the disclosure comprises a 21 nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense, wherein the antisense strand contains at least one thermally destabilizing nucleotide, where the at least one thermally destabilizing nucleotide occurs in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), wherein one end of the dsRNA is blunt, while the other end is comprises a 2 nt overhang, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2′-fluoro modifications; and (vii) the dsRNA comprises a blunt end at 5′-end of the antisense strand. Preferably, the 2 nt overhang is at the 3′-end of the antisense.

In some embodiments, every nucleotide in the sense strand and antisense strand of the dsRNA molecule may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases, the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

In some embodiments, each residue of the sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, or 2′-fluoro. The strands can contain more than one modification. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. It is to be understood that these modifications are in addition to the at least one thermally destabilizing modification of the duplex present in the antisense strand.

At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-deoxy, 2′-O-methyl, or 2′-fluoro modifications, acyclic nucleotides or others. In some embodiments, the sense strand and antisense strand each comprises two differently modified nucleotides selected from 2′-O-methyl or 2′-deoxy. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl nucleotide, 2′-deoxy nucleotide, 2′-deoxy-2′-fluoro nucleotide, 2′-O-N-methylacetamido (2′-O-NMA) nucleotide, a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide, 2′-O-aminopropyl (2′-O-AP) nucleotide, or 2′-ara-F nucleotide. Again, it is to be understood that these modifications are in addition to the at least one thermally destabilizing modification of the duplex present in the antisense strand.

In some embodiments, the dsRNA molecule of the disclosure comprises modifications of an alternating pattern, particular in the B1, B2, B3, B1′, B2′, B3′, B4′ regions. The term “alternating motif” or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In some embodiments, the dsRNA molecule of the disclosure comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 3′-5′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 3′-5′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

The dsRNA molecule of the disclosure may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.

In some embodiments, the dsRNA molecule comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region comprises two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3′-end of the antisense strand.

In some embodiments, the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, or 4 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the dsRNA molecule of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within positions 1-10 of the termini position(s) of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage at one end or both ends of the sense or antisense strand.

In some embodiments, the dsRNA molecule of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within positions 1-10 of the internal region of the duplex of each of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate methylphosphonate internucleotide linkage at position 8-16 of the duplex region counting from the 5′-end of the sense strand; the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modification within positions 1-10 of the termini position(s).

In some embodiments, the dsRNA molecule of the disclosure further comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 1-5 and one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 18-23 of the sense strand (counting from the 5′-end), and one to five phosphorothioate or methylphosphonate internucleotide linkage modification at positions 1 and 2 and one to five within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 (counting from the 5′-end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 (counting from the 5′-end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 20 and 21 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 21 and 22 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 22 and 23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 the antisense strand (counting from the 5′-end).

In some embodiments, compound of the disclosure comprises a pattern of backbone chiral centers. In some embodiments, a common pattern of backbone chiral centers comprises at least 5 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 6 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 7 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 8 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 9 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 16 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 19 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration, and no more than 8 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration, and no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration, and no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration, and no more than 4 internucleotidic linkages which are not chiral. In some embodiments, the internucleotidic linkages in the Sp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages which are not chiral are optionally contiguous or not contiguous.

In some embodiments, compound of the disclosure comprises a block is a stereochemistry block. In some embodiments, a block is an Rp block in that each internucleotidic linkage of the block is Rp. In some embodiments, a 5′-block is an Rp block. In some embodiments, a 3′-block is an Rp block. In some embodiments, a block is an Sp block in that each internucleotidic linkage of the block is Sp. In some embodiments, a 5′-block is an Sp block. In some embodiments, a 3′-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each internucleotidic linkage in a natural phosphate linkage.

In some embodiments, compound of the disclosure comprises a 5′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block comprises 4 or more nucleoside units. In some embodiments, a 5′-block comprises 5 or more nucleoside units. In some embodiments, a 5′-block comprises 6 or more nucleoside units. In some embodiments, a 5′-block comprises 7 or more nucleoside units. In some embodiments, a 3′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block comprises 4 or more nucleoside units. In some embodiments, a 3′-block comprises 5 or more nucleoside units. In some embodiments, a 3′-block comprises 6 or more nucleoside units. In some embodiments, a 3′-block comprises 7 or more nucleoside units.

In some embodiments, compound of the disclosure comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of internucleotidic linkage, e.g., natural phosphate linkage, modified internucleotidic linkage, Rp chiral internucleotidic linkage, Sp chiral internucleotidic linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

In some embodiments, the dsRNA molecule of the disclosure comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch can occur in the overhang region or the duplex region. The base pair can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In some embodiments, the dsRNA molecule of the disclosure comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In some embodiments, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

It was found that introducing 4′-modified or 5′-modified nucleotide to the 3′-end of a phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) linkage of a dinucleotide at any position of single stranded or double stranded oligonucleotide can exert steric effect to the internucleotide linkage and, hence, protecting or stabilizing it against nucleases.

In some embodiments, 5′-modified nucleoside is introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. For instance, a 5′-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The alkyl group at the 5′ position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 5′-alkylated nucleoside is 5′-methyl nucleoside. The 5′-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 4′-modified nucleoside is introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. For instance, a 4′-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The alkyl group at the 4′ position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4′-alkylated nucleoside is 4′-methyl nucleoside. The 4′-methyl can be either racemic or chirally pure R or S isomer. Alternatively, a 4′-O-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The 4′-O-alkyl of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The 4′-O-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 5′-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 5′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 5′-alkylated nucleoside is 5′-methyl nucleoside. The 5′-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 4′-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 4′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4′-alkylated nucleoside is 4′-methyl nucleoside. The 4′-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 4′-O-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 5′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The 4′-O-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, the dsRNA molecule of the disclosure can comprise 2′-5′ linkages (with 2′-H, 2′-OH, and 2′-OMe and with P═O or P═S). For example, the 2′-5′ linkages modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.

In another embodiment, the dsRNA molecule of the disclosure can comprise L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe). For example, these L sugars modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.

Various publications describe multimeric siRNA which can all be used with the dsRNA of the disclosure. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520 which are hereby incorporated by their entirely.

In some embodiments dsRNA molecules of the disclosure are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)₂(O)P—O-5′); 5′-diphosphate ((HO)₂(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, 5′-alkenylphosphonates (i.e. vinyl, substituted vinyl), (OH)₂(O)P-5′-CH₂-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-). In one example, the modification can in placed in the antisense strand of a dsRNA molecule.

Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.

Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In some embodiments, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

In some embodiments, a dsRNA of the disclosure is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXI)-(XXXIV):

wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C), T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C) are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C) are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C) are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) and L^(5C) represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a some embodiments, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a suitable pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In some embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

Redox Cleavable Linking Groups

In some embodiments, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

In some embodiments, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. In some embodiments, phosphate-based linking groups are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. In some embodiments, a phosphate-based linking group is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

Acid Cleavable Linking Groups

In some embodiments, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In some embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). In some embodiments, the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

Ester-Based Cleavable Linking Groups

In some embodiments, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

Peptide-Based Cleavable Linking Groups

In some embodiments, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide-based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above. Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which is herein incorporated by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an iRNA. The present disclosure also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds, or “chimeras,” in the context of the present disclosure, are iRNA compounds, e.g., dsRNAs, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

Delivery of iRNA

The delivery of an iRNA to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a composition comprising an iRNA, e.g. a dsRNA, to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

Direct Delivery

In general, any method of delivering a nucleic acid molecule can be adapted for use with an iRNA (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). However, there are three factors that are important to consider in order to successfully deliver an iRNA molecule in vivo: (a) biological stability of the delivered molecule, (2) preventing non-specific effects, and (3) accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example by direct injection or implantation into a tissue (as a non-limiting example, the eye) or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or that may degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya,Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo.

Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to other groups, e.g., a lipid or carbohydrate group as described herein. Such conjugates can be used to target iRNA to particular cells, e.g., liver cells, e.g., hepatocytes. For example, GalNAc conjugates or lipid (e.g., LNP) formulations can be used to target iRNA to particular cells, e.g., liver cells, e.g., hepatocytes.

iRNA molecules can also be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O., et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

Vector encoded iRNAs In another aspect, iRNA targeting VEGF-A can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively, each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In some embodiments, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

An iRNA expression vector is typically a DNA plasmid or viral vector. An expression vector compatible with eukaryotic cells, e.g., with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors contain convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

An iRNA expression plasmid can be transfected into a target cell as a complex with a cationic lipid carrier (e.g., Oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the disclosure. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.

Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.

Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.

In a specific embodiment, viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing an iRNA featured in the disclosure, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Use of Adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In some embodiments, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the disclosure, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol., 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

Another typical viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

III. Pharmaceutical Compositions Containing iRNA

In some embodiments, the disclosure provides pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the iRNA is useful for treating a disease or disorder related to the expression or activity of VEGF-A (e.g., an angiogenic ocular disorder). Such pharmaceutical compositions are formulated based on the mode of delivery. In some embodiments, compositions can be formulated for localized delivery, e.g., by intraocular delivery (e.g., intravitreal administration, e.g., intravitreal injection; transscleral administration, e.g., transscleral injection; subconjunctival administration, e.g., subconjunctival injection; retrobulbar administration, e.g., retrobulbar injection; intracameral administration, e.g., intracameral injection; or subretinal administration, e.g., subretinal injection). In other embodiments, compositions can be formulated for topical delivery. In another example, compositions can be formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. In some embodiments, a composition provided herein (e.g., a composition comprising a GalNAc conjugate or an LNP formulation) is formulated for intravenous delivery.

The pharmaceutical compositions featured herein are administered in a dosage sufficient to inhibit expression of VEGF-A. In general, a suitable dose of iRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day. The pharmaceutical composition may be administered once daily, or the iRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as can be used with the agents of the present disclosure. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

The effect of a single dose on VEGF-A levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5-day intervals, or at not more than 1, 2, 3, 4, 12, 24, or 36-week intervals.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the disclosure can be made using conventional methodologies or on the basis of in vivo testing using a suitable animal model.

A suitable animal model, e.g., a mouse or a cynomolgus monkey, e.g., an animal containing a transgene expressing human VEGF-A, can be used to determine the therapeutically effective dose and/or an effective dosage regimen administration of VEGF-A siRNA.

The present disclosure also includes pharmaceutical compositions and formulations that include the iRNA compounds featured herein. The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be local (e.g., by intraocular injection), topical (e.g., by an eye drop solution), or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal, or intraventricular administration.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the iRNAs featured in the disclosure are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the disclosure may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C120 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

Liposomal Formulations

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present disclosure, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

In order to traverse intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.). Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In some embodiments, a VEGF-A dsRNA featured in the disclosure is fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present disclosure typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present disclosure are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In some embodiments, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

In some embodiments, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.

In some embodiments, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG-distearyloxypropyl (C]₈). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

In some embodiments, the iRNA is formulated in a lipid nanoparticle (LNP).

LNP01

In some embodiments, the lipidoid ND984HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is herein incorporated by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (e.g., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are provided in the following table.

TABLE 6 Exemplary lipid formulations cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Cationic Lipid Lipid:siRNA ratio SNALP 1,2-Dilinolenyloxy-N,N - DLinDMA/DPPC/Cholesterol/PEG-cDMA dimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA~7:1 S-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DPPC/Cholesterol/PEG-cDMA [1,3]-dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMG di((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH- Lipid:siRNA 10:1 cyclopenta[d][1,3]dioxol-5-amine (ALN100) LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG 6,9,28,31-tetraen-19-yl 4- 50/10/38.5/1.5 (dimethylamino)butanoate (MC3) Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- C12-200/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2- 50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin- Lipid:siRNA 10:1 1-yl)ethylazanediyl)didodecan-2-ol (C12-200) LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG-DSG/GalNAc- PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC: distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.

XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. 61/185,712, filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, and International Application No. PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.

Synthesis of Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles featured in the disclosure may be prepared by known organic synthesis techniques. All substituents are as defined below unless indicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y), wherein n is 0, 1 or 2, R^(x) and R^(y) are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —OR^(x), heterocycle, —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y).

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods featured in the disclosure may require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this disclosure are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles featured in the disclosure are formulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.

Lipid A, where R₁ and R₂ are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R₃ and R₄ are independently lower alkyl or R₃ and R₄ can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:

Synthesis of 515

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0 OC under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO₃ solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H] −232.3 (96.94%).

Synthesis of 517A and 517B

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (— 3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO₃ (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50 mL). Organic phase was dried over an.Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS−[M+H] −266.3, [M+NH4+] −283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518

Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519

A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6, Found 654.6.

Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the disclosure are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the disclosure may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intravitreal, subretinal, transscleral, subconjunctival, retrobulbar, intracameral, intraventricular, or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations featured in the present disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions featured in the present disclosure may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Additional Formulations

Emulsions

The compositions of the present disclosure may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise, a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

In some embodiments of the present disclosure, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically, microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotopically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present disclosure will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.

Microemulsions of the present disclosure may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present disclosure. Penetration enhancers used in the microemulsions of the present disclosure may be classified as belonging to one of five broad categories--surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

Penetration Enhancers

In some embodiments, the present disclosure employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above-mentioned classes of penetration enhancers are described below in greater detail.

Surfactants: In connection with the present disclosure, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with the present disclosure, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present disclosure, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of β-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present disclosure. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invivogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.

Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

Certain compositions of the present disclosure also incorporate carrier compounds in the formulation. As used herein, “carrier compound” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Excipients

In contrast to a carrier compound, a pharmaceutical carrier or excipient may comprise, e.g., a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

The compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions, e.g., at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the disclosure include (a) one or more iRNA compounds and (b) one or more biologic agents which function by a non-RNAi mechanism. Examples of such biologic agents include agents that interfere with an interaction of VEGF-A and at least one VEGF-A binding partner.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are typical.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the disclosure lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured in the disclosure can be administered in combination with other known agents effective in treatment of diseases or disorders related to VEGF-A expression (e.g., an angiogenic ocular disorder). In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

Methods of Treating Disorders Related to Expression of VEGF-A

The present disclosure relates to the use of an iRNA targeting VEGF-A to inhibit VEGF-A expression and/or to treat a disease, disorder, or pathological process that is related to VEGF-A expression (e.g., an angiogenic ocular disorder).

In some aspects, a method of treatment of a disorder related to expression of VEGF-A is provided, the method comprising administering an iRNA (e.g., a dsRNA) disclosed herein to a subject in need thereof. In some embodiments, the iRNA inhibits (decreases) VEGF-A expression.

In some embodiments, the subject is an animal that serves as a model for a disorder related to VEGF-A expression, e.g., an angiogenic ocular disorder, e.g., AMD, DR, DME, RVO, MEfRVO, CVO, ROP, or mCNV.

Angiogenic Ocular Disorders

In some embodiments, the disorder related to VEGF-A expression is an angiogenic ocular disorder. Non-limiting examples of angiogenic ocular disorders that are treatable using the methods described herein include AMD (including wet AMD, exudative AMD, etc.), RVO (e.g., CRVO, MEfRVO, retinopathy of prematurity (ROP), or branch retinal vein occlusion (BRVO), DME, CNV (e.g., myopic CNV), iris neovascularization, neovascular glaucoma, post-surgical fibrosis in glaucoma, proliferative retinopathy, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreal neovascularization, pannus, pterygium, vascular retinopathy, von Hippel-Lindau disease, histoplasmosis, and diabetic retinopathies.

Clinical and pathological features of angiogenic ocular disorders include, but are not limited to, a reduction in visual acuity (e.g., characterized by floating spots, blurriness around the edges or center of field of vision (e.g., scotoma), metamorphopsia, and impaired color vision), increased leakage from the CNV, increased vascular permeability in the eye, collection of fluid or blood beneath the macula, abnormal ocular angiogenesis, and intraretinal hemorrhage.

In some embodiments, the subject with the angiogenic ocular disorder is less than 18 years old. In some embodiments, the subject with the angiogenic ocular disorder is an adult. In some embodiments, the subject has, or is identified as having, elevated levels of VEGF-A mRNA or protein relative to a reference level (e.g., a level of VEGF-A that is greater than a reference level).

In some embodiments, the angiogenic ocular disorder is diagnosed using analysis of a sample from the subject (e.g., an aqueous ocular fluid sample). In some embodiments, the sample is analyzed using a method selected from one or more of: fluorescent in situ hybridization (FISH), immunohistochemistry, VEGF-A immunoassay, electron microscopy, laser microdissection, and mass spectrometry. In some embodiments, angiogenic ocular disorder is diagnosed using any suitable diagnostic test or technique, e.g., angiography (e.g., fluorescein angiography or indocyanine green angiography), electroretinography, ultrasonography, pachymetry, optical coherence tomography (OCT), computed tomography (CT) and magnetic resonance imaging (MRI), tonometry, color vision testing, visual field testing, slit-lamp examination, ophthalmoscopy, and physical examination (e.g., to assess visual acuity (e.g., by fundoscopy or optical coherence tomography (OCT)).

Combination Therapies

In some embodiments, an iRNA (e.g., a dsRNA) disclosed herein is administered in combination with a second therapy (e.g., one or more additional therapies) known to be effective in treating a disorder related to VEGF-A expression (e.g., an angiogenic ocular disorder) or a symptom of such a disorder. The iRNA may be administered before, after, or concurrent with the second therapy. In some embodiments, the iRNA is administered before the second therapy. In some embodiments, the iRNA is administered after the second therapy. In some embodiments, the iRNA is administered concurrent with the second therapy.

The second therapy may be an additional therapeutic agent. The iRNA and the additional therapeutic agent can be administered in combination in the same composition or the additional therapeutic agent can be administered as part of a separate composition.

In some embodiments, the second therapy is a non-iRNA therapeutic agent that is effective to treat the disorder or symptoms of the disorder.

In some embodiments, the iRNA is administered in conjunction with a therapy.

Exemplary combination therapies include, but are not limited to, photodynamic therapy, photocoagulation therapy, a steroid, a non-steroidal anti-inflammatory agent, an anti-VEGF agent, and a vitrectomy.

In some embodiments, the anti-VEGF-A agent comprises a fusion protein. Exemplary anti-VEGF fusion proteins include, but are not limited to, aflibercept (EYLEA®). In some embodiments, the anti-VEGF-A fusion protein has the amino acid sequence of SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY TCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG (SEQ ID NO: 1905), or a variant thereof having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, the anti-VEGF-A agent is an antibody or antigen-binding fragment thereof (e.g., an anti-VEGF-A antibody molecule). Exemplary anti-VEGF-A antibody molecules include, but are not limited to, ranibizumab (LUCENTIS®) and brolucizumab (BEOVU®). In some embodiments, an anti-VEGF-A antibody molecule competes for binding to VEGF-A with ranibizumab or brolucizumab.

In some embodiments, the anti-VEGF-A antibody molecules comprises one or more (e.g., all three) of a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2) and a heavy chain complementarity determining region 3 (HCDR3). In some embodiments, the anti-VEGF-A antibody molecules comprises one or more (e.g., all three) of a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2) and a light chain complementarity determining region 3 (LCDR3).

In some embodiments, the anti-VEGF-A antibody molecule comprises a VH comprising one or more (e.g., all three) of a heavy chain complementarity determining region 1 (HCDR1) of an anti-VEGF-A antibody molecule described herein, e.g., in Table 7, (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a HCDR2 of an anti-VEGF-A antibody or antibody fragment thereof described herein, e.g., in Table 7, (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and a HCDR3 of an anti-VEGF-A antibody molecule described herein, e.g., in Table 7, (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the anti-VEGF-A antibody molecule comprises a VL comprising one or more (e.g., all three) of a light chain complementarity determining region 1 (LCDR1) of an anti-VEGF-A antibody molecule described herein, e.g., in Table 7, (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a LCDR2 of an anti-VEGF-A antibody molecule described herein, e.g., in Table 7, (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and a LCDR3 of an anti-VEGF-A antibody molecule described herein, e.g., in Table 7, (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the anti-VEGF-A antibody molecule comprises a VH comprising an amino acid sequence of an anti-VEGF-A antibody molecule described herein, e.g., in Table 7, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the anti-VEGF-A antibody molecule comprises a VL comprising the amino acid sequence of an anti-VEGF-A antibody molecule described herein, e.g., in Table 7, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, the anti-VEGF-A antibody molecule comprises a VH comprising the amino acid sequence of an anti-VEGF-A antibody molecule described herein, e.g., in Table 7, (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of an anti-VEGF-A antibody molecule described herein, e.g., in Table 7, (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In one embodiment, the anti-VEGF-A antibody molecule comprises a scFv comprising a light chain and a heavy chain of an amino acid sequence of anti-VEGF-A antibody molecule described herein, e.g., in Table 7. In one embodiment, the anti-VEGF-A antibody molecule (e.g., an scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region of an anti-VEGF-A antibody molecule described herein, e.g., in Table 7, or a sequence with 95-99% identity with an amino acid sequence of an anti-VEGF-A antibody molecule described herein, e.g., in Table 7; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region of an anti-VEGF-A antibody molecule described herein, e.g., in Table 7, or a sequence with 95-99% identity to an amino acid sequence of an anti-VEGF-A antibody molecule described herein, e.g., in Table 7.

In one embodiment, the anti-VEGF-A antibody molecule is a scFv, and a light chain variable region comprising an amino acid sequence of anti-VEGF-A antibody molecule described herein, e.g., in Table 7, is attached to a heavy chain variable region comprising an amino acid sequence of an anti-VEGF-A antibody molecule described herein, via a linker, e.g., a linker described herein. In one embodiment, the anti-VEGF-A antibody molecule includes a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO:1951). The light chain variable region and heavy chain variable region of a scFv can be, e.g., in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

TABLE 7 Exemplary Anti-VEGF Antibody Molecule Sequences Description SEQ ID NO. Sequence Brolucizumab Sequence 1906 MEIVMTQSPSTLSASVGDRVIITCQASEIIHSWL

WYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSG AEFTLTISSLQPDDFATYYCQNVYLASTNGAN

G QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQ LVESGGGLVQPGGSLRLSCTASGFSLTDYYYM

WVRQAPGKGLEWVGFIDPDDDPYYATWAKG

F TISRDNSKNTLYLQMNSLRAEDTAVYYCAGG

H NSGWGLDIWGQGTLVTVSS Brolucizumab VH 1907 EVQLVESGGGLVQPGGSLRLSCTASGFSLTDY

Y MTWVRQAPGKGLEWVGFIDPDDDPYYATWA

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAG GDHNSGWGLDIWGQGTLVTVSS Brolucizumab VL 1908 MEIVMTQSPSTLSASVGDRVIITCQASEIIHSWL

WYQQKPGKAPKLLIYLASTLASGVPSRFSGSG

G AEFTLTISSLQPDDFATYYCQNVYLASTNGAN

G QGTKLTVLG Brolucizumab Linker 1909 GGGGSGGGGSGGGGSGGGGS Ranibizumab Heavy Chain 1910 EVQLVESGGGLVQPGGSLRLSCAASGYDFT HYG MN WVRQAPGKGLEWVG WINTYTGEPTYAA

F KR RFTFSLDTSKSTAYLQMNSLRAEDTAVYY

A K YPYYYGTSHWYFDV WGOGTLVTVSSASTK

P SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP

S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT

L Ranibizumab Light Chain 1911 DIQLTQSPSSLSASVGDRVTITC SASQDISNYLN

YQQKPGKAPKVLIY FTSSLHS GVPSRFSGSGS

DFTLTISSLQPEDFATYYC QQYSTVPWT FGQG

KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL

N NFYPREAKVQWKVDNALQSGNSQESVTEQDS

DSTYSLSSTLTLSKADYEKHKVYACEVTHQGL

S PVTKSFNRGEC Ranibizumab VH 1912 EVQLVESGGGLVQPGGSLRLSCAASGYDFT HYG MN WVRQAPGKGLEWVG WINTYTGEPTYAA

F KR RFTFSLDTSKSTAYLQMNSLRAEDTAVYYCA K YPYYYGTSHWYFDV WGQGTLVT VSS Ranibizumab VL 1913 DIQLTQSPSSLSASVGDRVTITC SASQDISNYLN W YQQKPGKAPKVLIY FTSSLHS GVPSRFSGSGSGT DFTLTISSLQPEDFATYYC QQYSTVPWT FGQGT KVEIK Note: CDR sequences are bolded and underlined

indicates data missing or illegible when filed

Administration Dosages, Routes, and Timing

A subject (e.g., a human subject, e.g., a patient) can be administered a therapeutic amount of iRNA. The therapeutic amount can be, e.g., 0.05-50 mg/kg.

In some embodiments, the iRNA is formulated for delivery to a target organ, e.g., to the eye.

In some embodiments, the iRNA is formulated as a lipid formulation, e.g., an LNP formulation as described herein. In some such embodiments, the therapeutic amount is 0.05-5 mg/kg dsRNA. In some embodiments, the lipid formulation, e.g., LNP formulation, is administered intravenously.

In some embodiments, the iRNA is in the form of a GalNAc conjugate e.g., as described herein. In some such embodiments, the therapeutic amount is 0.5-50 mg dsRNA. In some embodiments, the e.g., GalNAc conjugate is administered subcutaneously.

In some embodiments, the administration is repeated, for example, on a regular basis, such as, daily, biweekly (i.e., every two weeks) for one month, two months, three months, four months, six months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer.

In some embodiments, the iRNA agent is administered in two or more doses. In some embodiments, the number or amount of subsequent doses is dependent on the achievement of a desired effect, e.g., to (a) inhibit angiogenesis; (b) inhibit or reduce the expression or activity of VEGF A; (c) inhibit choroidal neovascularization; (d) inhibit growth of new blood vessels in the choriocapillaris; (e) reduce retinal thickness; (f) increase visual acuity; or (g) reduce intraocular inflammation, or the achievement of a therapeutic or prophylactic effect, e.g., reduction or prevention of one or more symptoms associated with the disorder.

In some embodiments, the iRNA agent is administered according to a schedule. For example, the iRNA agent may be administered once per week, twice per week, three times per week, four times per week, or five times per week. In some embodiments, the schedule involves regularly spaced administrations, e.g., hourly, every four hours, every six hours, every eight hours, every twelve hours, daily, every 2 days, every 3 days, every 4 days, every 5 days, weekly, biweekly, or monthly. In some embodiments, the iRNA agent is administered at the frequency required to achieve a desired effect.

In some embodiments, the schedule involves closely spaced administrations followed by a longer period of time during which the agent is not administered. For example, the schedule may involve an initial set of doses that are administered in a relatively short period of time (e.g., about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, or about every 72 hours) followed by a longer time period (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks) during which the iRNA agent is not administered. In some embodiments, the iRNA agent is initially administered hourly and is later administered at a longer interval (e.g., daily, weekly, biweekly, or monthly). In some embodiments, the iRNA agent is initially administered daily and is later administered at a longer interval (e.g., weekly, biweekly, or monthly). In certain embodiments, the longer interval increases over time or is determined based on the achievement of a desired effect.

Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion dose, and monitored for adverse effects, such as an allergic reaction, or for elevated lipid levels or blood pressure. In another example, the patient can be monitored for unwanted effects.

Methods for Modulating Expression of VEGF-A

In some aspects, the disclosure provides a method for modulating (e.g., inhibiting or activating) the expression of VEGF-A, e.g., in a cell, in a tissue, or in a subject. In some embodiments, the cell or tissue is ex vivo, in vitro, or in vivo. In some embodiments, the cell or tissue is in the eye (e.g., retinal pigment epithelium (RPE), a retinal tissue, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, a photoreceptor cell, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel). In some embodiments, the cell or tissue is in a subject (e.g., a mammal, such as, for example, a human). In some embodiments, the subject (e.g., the human) is at risk, or is diagnosed with a disorder related to expression of VEGF-A expression, as described herein.

In some embodiments, the method includes contacting the cell with an iRNA as described herein, in an amount effective to decrease the expression of VEGF-A in the cell. In some embodiments, contacting a cell with an RNAi agent includes contacting a cell in vitro with the RNAi agent or contacting a cell in vivo with the RNAi agent. In some embodiments, the RNAi agent is put into physical contact with the cell by the individual performing the method, or the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell. Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, e.g., ocular tissue. For example, the RNAi agent may contain or be coupled to a ligand, e.g., a lipophilic moiety or moieties as described below and further detailed, e.g., in PCT/US2019/031170 which is incorporated herein by reference in its entirety, including the passages therein describing lipophilic moieties, that directs or otherwise stabilizes the RNAi agent at a site of interest. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.

The expression of VEGF-A may be assessed based on the level of expression of VEGF-A mRNA, VEGF-A protein, or the level of another parameter functionally linked to the level of expression of VEGF-A. In some embodiments, the expression of VEGF-A is inhibited by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, the iRNA has an IC50 in the range of 0.001-0.01 nM, 0.001-0.10 nM, 0.001-1.0 nM, 0.001-10 nM, 0.01-0.05 nM, 0.01-0.50 nM, 0.02-0.60 nM, 0.01-1.0 nM, 0.01-1.5 nM, 0.01-10 nM. The IC₅₀ value may be normalized relative to an appropriate control value, e.g., the IC₅₀ of a non-targeting iRNA.

In some embodiments, the method includes introducing into the cell or tissue an iRNA as described herein and maintaining the cell or tissue for a time sufficient to obtain degradation of the mRNA transcript of VEGF-A, thereby inhibiting the expression of VEGF-A in the cell or tissue.

In some embodiments, the method includes administering a composition described herein, e.g., a composition comprising an iRNA that binds VEGF-A, to the mammal such that expression of the target VEGF-A is decreased, such as for an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, or four weeks or longer. In some embodiments, the decrease in expression of VEGF-A is detectable within 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours of the first administration.

In some embodiments, the method includes administering a composition as described herein to a mammal such that expression of the target VEGF-A is increased by e.g., at least 10% compared to an untreated animal. In some embodiments, the activation of VEGF-A occurs over an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, four weeks, or more. Without wishing to be bound by theory, an iRNA can activate VEGF-A expression by stabilizing the VEGF-A mRNA transcript, interacting with a promoter in the genome, or inhibiting an inhibitor of VEGF-A expression.

The iRNAs useful for the methods and compositions featured in the disclosure specifically target RNAs (primary or processed) of VEGF-A. Compositions and methods for inhibiting the expression of VEGF-A using iRNAs can be prepared and performed as described elsewhere herein.

In some embodiments, the method includes administering a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of VEGF-A of the subject, e.g., the mammal, e.g., the human, to be treated. The composition may be administered by any appropriate means known in the art including, but not limited to ocular (e.g., intraocular), topical, and intravenous administration.

In certain embodiments, the composition is administered intraocularly (e.g., by intravitreal administration, e.g., intravitreal injection; transscleral administration, e.g., transscleral injection; subconjunctival administration, e.g., subconjunctival injection; retrobulbar administration, e.g., retrobulbar injection; intracameral administration, e.g., intracameral injection; or subretinal administration, e.g., subretinal injection. In other embodiments, the composition is administered topically. In other embodiments, the composition is administered by intravenous infusion or injection.

In certain embodiments, the composition is administered by intravenous infusion or injection. In some such embodiments, the composition comprises a lipid formulated siRNA (e.g., an LNP formulation, such as an LNP11 formulation) for intravenous infusion.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In the event of a discrepancy between the recited positions of the duplexes presented herein and the alignment of the duplexes to the recited sequences, the alignment of the duplexes to the recited sequence will govern. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Specific Embodiments

1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of vascular endothelial growth factor A (VEGF-A), wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of a coding strand of human VEGF-A and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of a non-coding strand of human VEGF-A such that the sense strand is complementary to the at least 15 contiguous nucleotides in the antisense strand.

2. The dsRNA agent of embodiment 1, wherein the coding strand of human VEGF-A comprises the sequence SEQ ID NO: 1.

3. The dsRNA agent of embodiment 1 or 2, wherein the non-coding strand of human VEGF-A comprises the sequence of SEQ ID NO: 2.

4. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of VEGF-A, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 15 contiguous nucleotides in the antisense strand.

5. The dsRNA agent of embodiment 4, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

6. The dsRNA of any of the preceding embodiments, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 17 contiguous nucleotides in the antisense strand.

7. The dsRNA of embodiment 6, wherein the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

8. The dsRNA of any of the preceding embodiments, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 19 contiguous nucleotides in the antisense strand.

9. The dsRNA of embodiment 8, wherein the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

10. The dsRNA of any of the preceding embodiments, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 21 contiguous nucleotides in the antisense strand.

11. The dsRNA of embodiment 10, wherein the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

12. The dsRNA agent of any one of embodiments 1-11, wherein the portion of the sense strand is a portion within nucleotides 1855-1875, 1858-1878, 2178-2198, 2181-2201, 2944-2964, 2946-2966, 2952-2972, 3361-3381, or 3362-3382 of SEQ ID NO: 1.

13. The dsRNA agent of any one of embodiments 1-12, wherein the portion of the sense strand is a portion within a sense strand from a duplex chosen from AD-1020574 (CGACAGAACAGUCCUUAAUCA (SEQ ID NO: 4200)), AD-901094 (CAGAACAGUCCUUAAUCCAGA (SEQ ID NO: 4201)), AD-1020575 (CAGAACAGUCCUUAAUCCAGA (SEQ ID NO: 4202)), AD-901100 (AACAGUGCUAAUGUUAUUGGA (SEQ ID NO: 4203)), AD-901101 (AGUGCUAAUGUUAUUGGUGUA (SEQ ID NO: 4204)), AD-901113 (GAGAAAGUGUUUUAUAUACGA (SEQ ID NO: 4205)), AD-901123 (AAAAUAGACAUUGCUAUUCUA (SEQ ID NO: 4206)), AD-901124 (AAAUAGACAUUGCUAUUCUGA (SEQ ID NO: 4207)), AD-901158 (GAAAGUGUUUUAUAUACGGUA (SEQ ID NO: 4208)), AD-901159 (GUUUUAUAUACGGUACUUAUA (SEQ ID NO: 4209)), AD-1020573 (AGUGCUAATGTUAUUGGUGUA (SEQ ID NO: 4210)), or AD-1023143 (AAAAUAGACATUGCUAUUCUA (SEQ ID NO: 4211)).

14. The dsRNA agent of any one of embodiments 1-13, wherein the portion of the sense strand is a sense strand chosen from the sense strands of AD-1020574 (CGACAGAACAGUCCUUAAUCA (SEQ ID NO: 4200)), AD-901094 (CAGAACAGUCCUUAAUCCAGA (SEQ ID NO: 4201)), AD-1020575 (CAGAACAGUCCUUAAUCCAGA (SEQ ID NO: 4202)), AD-901100 (AACAGUGCUAAUGUUAUUGGA (SEQ ID NO: 4203)), AD-901101 (AGUGCUAAUGUUAUUGGUGUA (SEQ ID NO: 4204)), AD-901113 (GAGAAAGUGUUUUAUAUACGA (SEQ ID NO: 4205)), AD-901123 (AAAAUAGACAUUGCUAUUCUA (SEQ ID NO: 4206)), AD-901124 (AAAUAGACAUUGCUAUUCUGA (SEQ ID NO: 4207)), AD-901158 (GAAAGUGUUUUAUAUACGGUA (SEQ ID NO: 4208)), AD-901159 (GUUUUAUAUACGGUACUUAUA (SEQ ID NO: 4209)), AD-1020573 (AGUGCUAATGTUAUUGGUGUA (SEQ ID NO: 4210)), or AD-1023143 (AAAAUAGACATUGCUAUUCUA (SEQ ID NO: 4211)).

15. The dsRNA of any one of embodiments 1-14, wherein the portion of the antisense strand is a portion within an antisense strand from a duplex chosen from AD-1020574 (UGAUUAAGGACUGUUCUGUCGAU (SEQ ID NO: 4212)), AD-901094 (UCUGGAUUAAGGACUGUUCUGUC (SEQ ID NO: 4213)), AD-1020575 (UCUGGATUAAGGACUGUUCUGUC (SEQ ID NO: 4214)), AD-901100 (UCCAAUAACAUUAGCACUGUUAA (SEQ ID NO: 4215)), AD-901101 (UACACCAAUAACAUUAGCACUGU (SEQ ID NO: 4216)), AD-901113 (UCGUAUAUAAAACACUUUCUCUU (SEQ ID NO: 4217)), AD-901123 (UAGAAUAGCAAUGUCUAUUUUAU (SEQ ID NO: 4218)), AD-901124 (UCAGAAUAGCAAUGUCUAUUUUA (SEQ ID NO: 4219)), AD-901158 (UACCGUAUAUAAAACACUUUCUC (SEQ ID NO: 4220)), AD-901159 (UAUAAGUACCGUAUAUAAAACAC (SEQ ID NO: 4221)), AD-1020573 (UACACCAAUAACATUAGCACUGU (SEQ ID NO: 4222)), or AD-1023143 (UAGAAUAGCAATGTCTAUUUUAU (SEQ ID NO: 4223)).

16. The dsRNA of any one of embodiments 1-15, wherein the portion of the antisense strand is an antisense strand chosen the antisense strands of AD-1020574 (UGAUUAAGGACUGUUCUGUCGAU (SEQ ID NO: 4212)), AD-901094 (UCUGGAUUAAGGACUGUUCUGUC (SEQ ID NO: 4213)), AD-1020575 (UCUGGATUAAGGACUGUUCUGUC (SEQ ID NO: 4214)), AD-901100 (UCCAAUAACAUUAGCACUGUUAA (SEQ ID NO: 4215)), AD-901101 (UACACCAAUAACAUUAGCACUGU (SEQ ID NO: 4216)), AD-901113 (UCGUAUAUAAAACACUUUCUCUU (SEQ ID NO: 4217)), AD-901123 (UAGAAUAGCAAUGUCUAUUUUAU (SEQ ID NO: 4218)), AD-901124 (UCAGAAUAGCAAUGUCUAUUUUA (SEQ ID NO: 4219)), AD-901158 (UACCGUAUAUAAAACACUUUCUC (SEQ ID NO: 4220)), AD-901159 (UAUAAGUACCGUAUAUAAAACAC (SEQ ID NO: 4221)), AD-1020573 (UACACCAAUAACATUAGCACUGU (SEQ ID NO: 4222)), or AD-1023143 (UAGAAUAGCAATGTCTAUUUUAU (SEQ ID NO: 4223)).

17. The dsRNA of any one of embodiments 1-16, wherein the sense strand and the antisense strand comprise nucleotide sequences of the paired sense strand and antisense strand of a duplex selected from AD-1020574 (SEQ ID NO: 4200 and 4212), AD-901094 (SEQ ID NO: 4201 and 4213), AD-1020575 (SEQ ID NO: 4202 and 4214), AD-901100 (SEQ ID NO: 4203 and 4215), AD-901101 (SEQ ID NO: 4204 and 4216), AD-901113 (SEQ ID NO: 4205 and 4217), AD-901123 (SEQ ID NO: 4206 and 4218), AD-901124 (SEQ ID NO: 4207 and 4219), AD-901158 (SEQ ID NO: 4208 and 4220), AD-901159 (SEQ ID NO: 4209 and 4221), AD-1020573 (SEQ ID NO: 4210 and 4222), or AD-1023143 (SEQ ID NO: 4211 and 4223).

18. The dsRNA agent of any one of embodiments 1-11, wherein the portion of the sense strand is a portion within a sense strand from a duplex chosen from AD-953374 (SEQ ID NO: 813), AD-953504 (SEQ ID NO: 1297), AD-953481 (SEQ ID NO: 1298), AD-953351 (SEQ ID NO: 800), AD-901356 (SEQ ID NO: 261), AD-953344 (SEQ ID NO: 787), AD-901355 (SEQ ID NO: 262), AD-953410 (SEQ ID NO: 845), AD-953363 (SEQ ID NO: 779), AD-953411 (SEQ ID NO: 844), AD-953350 (SEQ ID NO: 784), or AD-953375 (SEQ ID NO: 790).

19. The dsRNA agent of any one of embodiments 1-11 or 18, wherein the portion of the sense strand is a sense strand chosen from the sense strands of AD-953374 (SEQ ID NO: 813), AD-953504 (SEQ ID NO: 1297), AD-953481 (SEQ ID NO: 1298), AD-953351 (SEQ ID NO: 800), AD-901356 (SEQ ID NO: 261), AD-953344 (SEQ ID NO: 787), AD-901355 (SEQ ID NO: 262), AD-953410 (SEQ ID NO: 845), AD-953363 (SEQ ID NO: 779), AD-953411 (SEQ ID NO: 844), AD-953350 (SEQ ID NO: 784), or AD-953375 (SEQ ID NO: 790).

20. The dsRNA of any one of embodiments 1-11 or 18-19, wherein the portion of the antisense strand is a portion within an antisense strand from a duplex chosen from AD-953374 (SEQ ID NO: 943), AD-953504 (SEQ ID NO: 1427), AD-953481 (SEQ ID NO: 1428), AD-953351 (SEQ ID NO: 930), AD-901356 (SEQ ID NO: 390), AD-953344 (SEQ ID NO: 917), AD-901355 (SEQ ID NO: 391), AD-953410 (SEQ ID NO: 975), AD-953363 (SEQ ID NO: 909), AD-953411 (SEQ ID NO: 974), AD-953350 (SEQ ID NO: 914), or AD-953375 (SEQ ID NO: 920).

21. The dsRNA of any one of embodiments 1-11 or 18-20, wherein the portion of the antisense strand is an antisense strand chosen from AD-953374 (SEQ ID NO: 943), AD-953504 (SEQ ID NO: 1427), AD-953481 (SEQ ID NO: 1428), AD-953351 (SEQ ID NO: 930), AD-901356 (SEQ ID NO: 390), AD-953344 (SEQ ID NO: 917), AD-901355 (SEQ ID NO: 391), AD-953410 (SEQ ID NO: 975), AD-953363 (SEQ ID NO: 909), AD-953411 (SEQ ID NO: 974), AD-953350 (SEQ ID NO: 914), or AD-953375 (SEQ ID NO: 920).

22. The dsRNA of any one of embodiments 1-11, or 18-21 wherein the sense strand and the antisense strand of comprise the nucleotide sequences of the paired sense strand and antisense strand of a duplex selected from AD-953374 (SEQ ID NO: 813 and 943), AD-953504 (SEQ ID NO: 1297 and 1427), AD-953481 (SEQ ID NO: 1298 and 1428), AD-953351 (SEQ ID NO: 800 and 930), AD-901356 (SEQ ID NO: 261 and 390), AD-953344 (SEQ ID NO: 787 and 917), AD-901355 (SEQ ID NO: 262 and 391), AD-953410 (SEQ ID NO: 845 and 975), AD-953363 (SEQ ID NO: 779 and 909), AD-953411 (SEQ ID NO: 844 and 974), AD-953350 (SEQ ID NO: 784 and 914), or AD-953375 (SEQ ID NO: 790 and 920).

23. The dsRNA agent of any one of the preceding embodiments, wherein the portion of the sense strand is a portion within a sense strand in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B.

24. The dsRNA agent of any one of the preceding embodiments, wherein the portion of the antisense strand is a portion within an antisense strand in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B.

25. The dsRNA agent of any of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B.

26. The dsRNA agent of any of the preceding embodiments, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B that corresponds to the antisense sequence.

27. The dsRNA agent of any of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B.

28. The dsRNA agent of any of the preceding embodiments, wherein the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B that corresponds to the antisense sequence.

29. The dsRNA agent of any of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0,1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B.

30. The dsRNA agent of any of the preceding embodiments, wherein the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B that corresponds to the antisense sequence.

31. The dsRNA agent of any of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0,1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B.

32. The dsRNA agent of any of the preceding embodiments, wherein the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B that corresponds to the antisense sequence.

33. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of VEGF-A, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of an antisense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B, and the sense strand comprises a nucleotide sequence of a sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A and 18B that corresponds to the antisense sequence.

34. The dsRNA agent of embodiment 33, wherein the antisense strand comprises a nucleotide sequence of an antisense sequence listed in Table 2A, and the sense strand comprises a nucleotide sequence of a sense sequence listed in Table 2A that corresponds to the antisense sequence.

35. The dsRNA agent of embodiment 33, wherein the antisense strand comprises a nucleotide sequence of an antisense sequence listed in Table 3A, and the sense strand comprises a nucleotide sequence of a sense sequence listed in Table 3A that corresponds to the antisense sequence.

36. The dsRNA agent of embodiment 33, wherein the antisense strand comprises a nucleotide sequence of an antisense sequence listed in Table 4A, and the sense strand comprises a nucleotide sequence of a sense sequence listed in Table 4A that corresponds to the antisense sequence.

37. The dsRNA agent of embodiment 33, wherein the antisense strand comprises a nucleotide sequence of an antisense sequence listed in Table 18A, and the sense strand comprises a nucleotide sequence of a sense sequence listed in Table 18A that corresponds to the antisense sequence.

38. The dsRNA agent of any one of embodiments 33 or 37, wherein the dsRNA agent is AD-1020574, AD-901094, AD-1020575, AD-901100, AD-901101, AD-901113, AD-901123, AD-901124, AD-901158, AD-901159, AD-1020573, or AD-1023143.

39. The dsRNA agent of any one of embodiments 33 or 37-38, comprising:

(i) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 4164, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4176;

(ii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1465, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4177;

(iii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1466, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4178;

(iv) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1467, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4179;

(v) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1468, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4180;

(vi) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1469, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4181;

(vii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1470, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4182;

(viii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1471, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4183;

(ix) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1472, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4184;

(x) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1473, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4185;

(xi) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1474, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4186; or

(xii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1475, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4187.

40. The dsRNA agent of any one of embodiments 33-36, wherein the dsRNA agent is AD-953374, AD-953504, AD-953481, AD-953351, AD-901356, AD-953344, AD-901355, AD-953410, AD-953363, AD-953411, AD-953350, or AD-953375.

41. The dsRNA agent of any one of embodiments 33-36 or 40, comprising:

(i) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 553, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 683;

(ii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1037, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 1167;

(iii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1038, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 1168;

(iv) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 540, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 670;

(v) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 3, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 132;

(vi) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 527, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 657;

(vii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 4, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 133;

(viii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 585, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 715;

(ix) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 519, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 649;

(x) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 584, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 714;

(xi) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 524, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 654; or

(xii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 530, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 660.

42. The dsRNA agent of any of the preceding embodiments, wherein the sense strand is at least 23 nucleotides in length, e.g., 23-30 nucleotides in length.

43. The dsRNA agent of any of the preceding embodiments, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties.

44. The dsRNA agent of embodiment 43, wherein the lipophilic moiety is conjugated to one or more positions in the double stranded region of the dsRNA agent.

45. The dsRNA agent of embodiment 43 or 44, wherein the lipophilic moiety is conjugated via a linker or carrier.

46. The dsRNA agent of any one of embodiments 43-45, wherein lipophilicity of the lipophilic moiety, measured by log Kow, exceeds 0.

47. The dsRNA agent of any one of the preceding embodiments, wherein the hydrophobicity of the double-stranded RNAi agent, measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent, exceeds 0.2.

48. The dsRNA agent of embodiment 47, wherein the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

49. The dsRNA agent of any of the preceding embodiments, wherein the dsRNA agent comprises at least one modified nucleotide.

50. The dsRNA agent of embodiment 49, wherein no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand are unmodified nucleotides.

51. The dsRNA agent of embodiment 50, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

52. The dsRNA agent of any one of embodiments 49-51, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′-phosphate mimic, a glycol modified nucleotide, and a 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof.

53. The dsRNA agent of any of embodiments 49-51, wherein no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand include modifications other than 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA).

54. The dsRNA agent of any of the preceding embodiments, which comprises a non-nucleotide spacer (wherein optionally the non-nucleotide spacer comprises a C3-C6 alkyl) between two of the contiguous nucleotides of the sense strand or between two of the contiguous nucleotides of the antisense strand.

55. The dsRNA agent of any of the preceding embodiments, wherein each strand is no more than 30 nucleotides in length.

56. The dsRNA agent of any of the preceding embodiments, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide.

57. The dsRNA agent of any of the preceding embodiments, wherein at least one strand comprises a 3′ overhang of at least 2 nucleotides.

58. The dsRNA agent of any of the preceding embodiments, wherein the double stranded region is 15-30 nucleotide pairs in length.

59. The dsRNA agent of embodiment 58, wherein the double stranded region is 17-23 nucleotide pairs in length.

60. The dsRNA agent of embodiment 58, wherein the double stranded region is 17-25 nucleotide pairs in length.

61. The dsRNA agent of embodiment 58, wherein the double stranded region is 23-27 nucleotide pairs in length.

62. The dsRNA agent of embodiment 58, wherein the double stranded region is 19-21 nucleotide pairs in length.

63. The dsRNA agent of embodiment 58, wherein the double stranded region is 21-23 nucleotide pairs in length.

64. The dsRNA agent of any of the preceding embodiments, wherein each strand has 19-30 nucleotides.

65. The dsRNA agent of any of the preceding embodiments, wherein each strand has 19-23 nucleotides.

66. The dsRNA agent of any of the preceding embodiments, wherein each strand has 21-23 nucleotides.

67. The dsRNA agent of any of the preceding embodiments, wherein the agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

68. The dsRNA agent of embodiment 67, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand.

69. The dsRNA agent of embodiment 68, wherein the strand is the antisense strand.

70. The dsRNA agent of embodiment 68, wherein the strand is the sense strand.

71. The dsRNA agent of embodiment 67, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand.

72. The dsRNA agent of embodiment 71, wherein the strand is the antisense strand.

73. The dsRNA agent of embodiment 71, wherein the strand is the sense strand.

74. The dsRNA agent of embodiment 67, wherein each of the 5′- and 3′-terminus of one strand comprises a phosphorothioate or methylphosphonate internucleotide linkage.

75. The dsRNA agent of embodiment 74, wherein the strand is the antisense strand.

76. The dsRNA agent of any of the preceding embodiments, wherein the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

77. The dsRNA agent of embodiment 74, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

78. The dsRNA agent of any one of embodiments 43-77, wherein one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand.

79. The dsRNA agent of embodiment 78, wherein the one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand via a linker or carrier.

80. The dsRNA agent of embodiment 79, wherein the internal positions include all positions except the terminal two positions from each end of the at least one strand.

81. The dsRNA agent of embodiment 79, wherein the internal positions include all positions except the terminal three positions from each end of the at least one strand.

82. The dsRNA agent of any one of embodiments 79-61, wherein the internal positions exclude a cleavage site region of the sense strand.

83. The dsRNA agent of embodiment 82, wherein the internal positions include all positions except positions 9-12, counting from the 5′-end of the sense strand.

84. The dsRNA agent of embodiment 82, wherein the internal positions include all positions except positions 11-13, counting from the 3′-end of the sense strand.

85. The dsRNA agent of any one of embodiments 79-81, wherein the internal positions exclude a cleavage site region of the antisense strand.

86. The dsRNA agent of embodiment 85, wherein the internal positions include all positions except positions 12-14, counting from the 5′-end of the antisense strand.

87. The dsRNA agent of any one of embodiments 79-81, wherein the internal positions include all positions except positions 11-13 on the sense strand, counting from the 3′-end, and positions 12-14 on the antisense strand, counting from the 5′-end.

88. The dsRNA agent of any one of embodiments 43-87, wherein the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5′end of each strand.

89. The dsRNA agent of embodiment 88, wherein the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5′-end of each strand.

90. The dsRNA agent of embodiment 44, wherein the positions in the double stranded region exclude a cleavage site region of the sense strand.

91. The dsRNA agent of any one of embodiments 43-90, wherein the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand.

92. The dsRNA agent of embodiment 91, wherein the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand.

93. The dsRNA agent of embodiment 91, wherein the lipophilic moiety is conjugated to position 21, position 20, or position 15 of the sense strand.

94. The dsRNA agent of embodiment 91, wherein the lipophilic moiety is conjugated to position 20 or position 15 of the sense strand.

95. The dsRNA agent of embodiment 91, wherein the lipophilic moiety is conjugated to position 16 of the antisense strand.

96. The dsRNA agent of embodiment 91, wherein the lipophilic moiety is conjugated to position 6, counting from the 5′-end of the sense strand.

97. The dsRNA agent of any one of embodiments 43-96, wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

98. The dsRNA agent of embodiment 98, wherein the lipophilic moiety is selected from the group consisting of lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

99. The dsRNA agent of embodiment 98, wherein the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

100. The dsRNA agent of embodiment 99, wherein the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain.

101. The dsRNA agent of embodiment 99, wherein the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

102. The dsRNA agent of any one of embodiments 43-101, wherein the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) or the double stranded region.

103. The dsRNA agent of embodiment 102, wherein the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.

104. The dsRNA agent of any one of embodiments 43-101, wherein the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.

105. The double-stranded iRNA agent of any one of embodiments 43-104, wherein the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.

106. The dsRNA agent of any one of embodiments 43-105, wherein the lipophilic moiety is conjugated via a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

107. The dsRNA agent of any one of embodiments 43-106, wherein the 3′ end of the sense strand is protected via an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

108. The dsRNA agent of any one of embodiments 43-107, further comprising a targeting ligand, e.g., a ligand that targets an ocular tissue or a liver tissue.

109. The dsRNA agent of embodiment 108, wherein the ligand is conjugated to the sense strand.

110. The dsRNA agent of embodiment 108 or 109, wherein the ligand is conjugated to the 3′ end or the 5′ end of the sense strand.

111. The dsRNA agent of embodiment 108 or 109, wherein the ligand is conjugated to the 3′ end of the sense strand.

112. The dsRNA agent of any one of embodiments 108-111, wherein the ocular tissue is a retinal pigment epithelium (RPE) or choroid tissue, e.g., a choroid vessel.

113. The dsRNA agent of any one of embodiments 108-111, wherein the targeting ligand comprises N-acetylgalactosamine (GalNAc).

114. The dsRNA agent of any one of embodiments 108-111, wherein the targeting ligand is one or more GalNAc conjugates or one or more or GalNAc derivatives.

115. The dsRNA agent of embodiment 114, wherein the one or more GalNAc conjugates or one or more GalNAc derivatives are attached through a monovalent linker, or a bivalent, trivalent, or tetravalent branched linker.

116. The dsRNA agent of embodiment 114, wherein the ligand is

117. The dsRNA agent of embodiment 116, wherein the dsRNA agent is conjugated to the ligand as shown in the following schematic

wherein X is O or S.

118. The dsRNA agent of embodiment 117, wherein the X is O.

119. The dsRNA agent of any one of embodiments 1-118, further comprising a terminal, chiral modification occurring at the first internucleotide linkage at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.

120. The dsRNA agent of any one of embodiments 1-118, further comprising

a terminal, chiral modification occurring at the first and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

121. The dsRNA agent of any one of embodiments 1-118, further comprising

a terminal, chiral modification occurring at the first, second and third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

122. The dsRNA agent of any one of embodiments 1-118, further comprising

a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

a terminal, chiral modification occurring at the third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration,

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

123. The dsRNA agent of any one of embodiments 1-118, further comprising

a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

124. The dsRNA agent of any one of embodiments 1-123, further comprising a phosphate or phosphate mimic at the 5′-end of the antisense strand.

125. The dsRNA agent of embodiment 104, wherein the phosphate mimic is a 5′-vinyl phosphonate (VP).

126. A cell containing the dsRNA agent of any one of embodiments 1-125.

127. A human ocular cell, e.g., (an RPE cell, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, or a photoreceptor cell) comprising a reduced level of VEGF-A mRNA or a level of VEGF-A protein as compared to an otherwise similar untreated cell, wherein optionally the level is reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

128. The human cell of embodiment 127, which was produced by a process comprising contacting a human cell with the dsRNA agent of any one of embodiments 1-125.

129. A pharmaceutical composition for inhibiting expression of VEGF-A, comprising the dsRNA agent of any one of embodiments 1-125.

130. A pharmaceutical composition comprising the dsRNA agent of any one of embodiments 1-125 and a lipid formulation.

131. A method of inhibiting expression of VEGF-A in a cell, the method comprising:

-   -   (a) contacting the cell with the dsRNA agent of any one of         embodiments 1-125, or a pharmaceutical composition of embodiment         129 or 130; and     -   (b) maintaining the cell produced in step (a) for a time         sufficient to obtain degradation of the mRNA transcript of         VEGF-A, thereby inhibiting expression of VEGF-A in the cell.

132. A method of inhibiting expression of VEGF-A in a cell, the method comprising:

-   -   (a) contacting the cell with the dsRNA agent of any one of         embodiments 1-125, or a pharmaceutical composition of embodiment         129 or 130; and     -   (b) maintaining the cell produced in step (a) for a time         sufficient to reduce levels of VEGF-A mRNA, VEGF-A protein, or         both of VEGF-A mRNA and protein, thereby inhibiting expression         of VEGF-A in the cell.

133. The method of embodiment 131 or 132, wherein the cell is within a subject.

134. The method of embodiment 133, wherein the subject is a human.

135. The method of any one of embodiments 131-134, wherein the level of VEGF-A mRNA is inhibited by at least 50%.

136. The method of any one of embodiments 131-134, wherein the level of VEGF-A protein is inhibited by at least 50%.

137. The method of embodiment 134-136, wherein inhibiting expression of VEGF-A decreases a VEGF-A protein level in a biological sample (e.g., an aqueous ocular fluid sample) from the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

138. The method of any one of embodiments 134-137, wherein the subject has been diagnosed with a VEGF-A-associated disorder, e.g., wet age-related macular degeneration (wet AMD), diabetic retinopathy (DR), diabetic macular edema (DME), retinal vein occlusion (RVO), macular edema following retinal vein occlusion (MEfRVO), retinopathy of prematurity (ROP), or myopic choroidal neovascularization (mCNV).

139. A method of inhibiting expression of VEGF-A in an ocular cell or tissue, the method comprising:

-   -   (a) contacting the cell or tissue with a dsRNA agent that binds         VEGF-A; and     -   (b) maintaining the cell or tissue produced in step (a) for a         time sufficient to reduce levels of VEGF-A mRNA, VEGF-A protein,         or both of VEGF-A mRNA and protein, thereby inhibiting         expression of VEGF-A in the cell or tissue.

140. The method of embodiment 139, wherein the ocular cell or tissue comprises an RPE cell, a retinal tissue, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, a photoreceptor cell, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel.

141. A method of treating a subject diagnosed with a VEGF-A-associated disorder comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of embodiments 1-125 or a pharmaceutical composition of embodiment 129 or 130, thereby treating the disorder.

142. The method of embodiment 138 or 141, wherein the VEGF-A-associated disorder is an angiogenic ocular disorder.

143. The method of embodiment 142, wherein the angiogenic ocular disorder is selected from the group consisting of AMD, DR, DME, RVO, CVO, MEfRVO, ROP, or mCNV.

144. The method of any one of embodiments 141-143, wherein treating comprises amelioration of at least one sign or symptom of the disorder.

145. The method of embodiment 144, wherein at least one sign or symptom of the angiogenic ocular disorder comprises a measure of one or more of angiogenesis, choroidal neovascularization, ocular inflammation, visual acuity, or presence, level, or activity of VEGF-A (e.g., VEGF-A gene, VEGF-A mRNA, or VEGF-A protein).

146. The method of any one of embodiments 141-143, where treating comprises prevention of progression of the disorder.

147. The method of any one of embodiments 144-146, wherein the treating comprises one or more of (a) inhibiting angiogenesis; (b) inhibiting or reducing the expression or activity of VEGF-A; (c) inhibiting choroidal neovascularization; (d) inhibiting growth of new blood vessels in the choriocapillaris; (e) reducing retinal thickness; (f) increasing visual acuity; or (g) reducing intraocular inflammation.

148. The method of embodiment 147, wherein the treating results in at least a 30% mean reduction from baseline of VEGF mRNA in the retina, RPE, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel.

149. The method of embodiment 148 wherein the treating results in at least a 60% mean reduction from baseline of VEGF mRNA in the retina, RPE, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel.

150. The method of embodiment 149, wherein the treating results in at least a 90% mean reduction from baseline of VEGF mRNA in the retina, RPE, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel.

151. The method of any one of embodiments 144-149, wherein after treatment the subject experiences at least an 8-week duration of knockdown following a single dose of dsRNA as assessed by VEGF-A protein in the retina.

152. The method of embodiment 151, wherein treating results in at least a 12-week duration of knockdown following a single dose of dsRNA as assessed by VEGF-A protein in the retina.

153. The method of embodiment 152, wherein treating results in at least a 16-week duration of knockdown following a single dose of dsRNA as assessed by VEGF-A protein in the retina.

154. The method of any of embodiments 133-153, wherein the subject is human.

155. The method of any one of embodiments 134-154, wherein the dsRNA agent is administered at a dose of about 0.01 mg/kg to about 50 mg/kg.

156. The method of any one of embodiments 134-155, wherein the dsRNA agent is administered to the subject intraocularly, intravenously, or topically.

157. The method of embodiment 156, wherein the intraocular administration comprises intravitreal administration (e.g., intravitreal injection), transscleral administration (e.g., transscleral injection), subconjunctival administration (e.g., subconjunctival injection), retrobulbar administration (e.g., retrobulbar injection), intracameral administration (e.g., intracameral injection), or subretinal administration (e.g., subretinal injection).

158. The method of any one of embodiments 134-157, further comprising measuring level of VEGF-A (e.g., VEGF-A gene, VEGF-A mRNA, or VEGF-A protein) in the subject.

159. The method of embodiment 158, where measuring the level of VEGF-A in the subject comprises measuring the level of VEGF-A gene, VEGF-A protein or VEGF-A mRNA in a biological sample from the subject (e.g., an aqueous ocular fluid sample).

160. The method of any one of embodiments 134-159, further comprising performing a blood test, an imaging test, or an aqueous ocular fluid biopsy.

161. The method of any one of embodiments 158-168, wherein measuring level of VEGF-A (e.g., VEGF-A gene, VEGF-A mRNA, or VEGF-A protein) in the subject is performed prior to treatment with the dsRNA agent or the pharmaceutical composition.

162. The method of embodiment 161, wherein, upon determination that a subject has a level of VEGF-A (e.g., VEGF-A gene, VEGF-A mRNA, or VEGF-A protein) that is greater than a reference level, the dsRNA agent or the pharmaceutical composition is administered to the subject.

163. The method of any one of embodiments 159-162, wherein measuring level of VEGF-A (e.g., VEGF-A gene, VEGF-A mRNA, or VEGF-A protein) in the subject is performed after treatment with the dsRNA agent or the pharmaceutical composition.

164. The method of any one of embodiments 141-163, further comprising administering to the subject an additional agent and/or therapy suitable for treatment or prevention of an VEGF-A-associated disorder.

165. The method of embodiment 164, wherein the additional agent and/or therapy comprises one or more of a photodynamic therapy, photocoagulation therapy, a steroid, a non-steroidal anti-inflammatory agent, an anti-VEGF-A agent, and/or a vitrectomy.

166. The method of embodiment 165, wherein the anti-VEGF-A agent is a fusion protein or an anti-VEGF-A antibody or antigen-binding fragment thereof (e.g., an anti-VEGF-A antibody molecule).

EXAMPLES Example 1. VEGF-A siRNA

Nucleic acid sequences provided herein are represented using standard nomenclature. See the abbreviations of Table 1.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acid sequence representation It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) A Adenosine-3′-phosphate Ab beta-L-adenosine-3′-phosphate Abs beta-L-adenosine-3′-phosphorothioate Af 2′-fluoroadenosine-3′-phosphate Afs 2′-fluoroadenosine-3′-phosphorothioate (Ahd) 2′-O-hexadecyl-adenosine-3′-phosphate (Ahds) 2′-O-hexadecyl-adenosine-3′-phosphorothioate As adenosine-3′-phosphorothioate (A2p) adenosine 2′-phosphate C cytidine-3′-phosphate Cb beta-L-cytidine-3′-phosphate Cbs beta-L-cytidine-3′-phosphorothioate Cf 2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioate (Chd) 2’-O-hexadecyl-cytidine-3’-phosphate (Chds) 2’-O-hexadecyl-cytidine-3’-phosphorothioate Cs cytidine-3′-phosphorothioate (C2p) cytosine 2′-phosphate G guanosine-3′-phosphate Gb beta-L-guanosine-3′-phosphate Gbs beta-L-guanosine-3′-phosphorothioate Gf 2′-fluoroguanosine-3′-phosphate Gfs 2′-fluoroguanosine-3′-phosphorothioate (Ghd) 2′-O-hexadecyl-guanosine-3′-phosphate (Ghds) 2′-O-hexadecyl-guanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioate T 5′-methyluridine-3′-phosphate Tb beta-L-thymidine-3′-phosphate Tbs beta-L-thymidine-3′-phosphorothioate Tf 2′-fluoro-5-methyluridine-3′-phosphate Tfs 2′-fluoro-5-methyluridine-3′-phosphorothioate Tgn thymidine-glycol nucleic acid (GNA) S-Isomer Agn adenosine-glycol nucleic acid (GNA) S-Isomer Cgn cytidine-glycol nucleic acid (GNA) S-Isomer Ggn guanosine-glycol nucleic acid (GNA) S-Isomer Ts 5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Ub beta-L-uridine-3′-phosphate Ubs beta-L-uridine-3′-phosphorothioate Uf 2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioate (Uhd) 2′-O-hexadecyl-uridine-3′-phosphate (Uhds) 2′-O-hexadecyl-uridine-3′-phosphorothioate Us uridine-3′-phosphorothioate (U2p) uracil 2′-phosphate N any nucleotide (G, A, C, T or U) VP Vinyl phosphonate a 2′-O-methyladenosine-3′-phosphate as 2′-O-methyladenosine-3′-phosphorothioate c 2′-O-methylcytidine-3′-phosphate cs 2′-O-methylcytidine-3′-phosphorothioate g 2′-O-methylguanosine-3′-phosphate gs 2′-O-methylguanosine-3′-phosphorothioate t 2′-O-methyl-5-methyluridine-3′-phosphate ts 2′-O-methyl-5-methyluridine-3′-phosphorothioate u 2′-O-methyluridine-3′-phosphate us 2′-O-methyluridine-3′-phosphorothioate dA 2′-deoxyadenosine-3′-phosphate dAs 2′-deoxyadenosine-3′-phosphorothioate dC 2′-deoxycytidine-3′-phosphate dCs 2′-deoxycytidine-3′-phosphorothioate dG 2′-deoxyguanosine-3′-phosphate dGs 2′-deoxyguanosine-3′-phosphorothioate dT 2′-deoxythymidine dTs 2′-deoxythymidine-3′-phosphorothioate dU 2′-deoxyuridine s phosphorothioate linkage L96¹ N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol Hyp-(GalNAc-alkyl)3 (Aeo) 2′-O-methoxyethyladenosine-3′-phosphate (Aeos) 2′-O-methoxyethyladenosine-3′-phosphorothioate (Geo) 2′-O-methoxyethylguanosine-3′-phosphate (Geos) 2′-O-methoxyethylguanosine-3′-phosphorothioate (Teo) 2′-O-methoxyethyl-5-methyluridine-3′-phosphate (Teos) 2′-O-methoxyethyl-5-methyluridine-3′-phosphorothioate (m5Ceo) 2′-O-methoxyethyl-5-methylcytidine-3′-phosphate (m5Ceos) 2′-O-methoxyethyl-5-methylcytidine-3′-phosphorothioate ¹The chemical structure of L96 is as follows:

Experimental Methods Bioinformatics

Transcripts

Three sets of siRNAs targeting the human VEGF-A, “vascular endothelial growth factor A” (human: NCBI refseqID NM_001171623; NCBI GeneID: 7422) were generated. The human NM_001171623 REFSEQ mRNA, version 1, has a length of 3677 bases. Pairs of oligos were generated using bioinformatic methods and ranked, and exemplary pairs of oligos are shown in Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 8A, Table 8B, Table 10A, Table 10B, Table 18A, and Table 18B. Modified sequences are presented in Table 2A, Table 3A, Table 4A, Table 8A, Table 10A, Table 18A. Unmodified sequences are presented in Table 2B, Table 3B, Table 4B, Table 8B, Table 10B, Table 18B.

Similarly, a set of siRNAs targeting rat VEGF-A (rat: NCBI refseqID NM_001110333; NCBI GeneID 83785 were generated. The rat NM_001110333.2REFSEQ mRNA, version 2, has a length of 3474 base pairs. Pairs of oligos were generated using bioinformatic methods and ranked, and exemplary pairs of oligos are shown in Table 5A and Table 5B. Modified sequences are presented in Table 5A. Unmodified sequences are presented in Table 5B.

TABLE 2A Exemplary Human VEGF-A siRNA Modified Single Strands and Duplex Sequences SEQ Anti- ID SEQ ID Sense SEQ sense NO: NO: Duplex Oligo ID NO: Oligo (Anti- mRNA target (mRNA Name Name (Sense) Sense Sequence Name sense) Antisense Sequence sequence target) AD- A- 4156 asasgac(Uhd)GfaUfAfCfag A- 130 VPusGfsaucg(Tgn)ucu GAAAGACUGAUA 4224 901349.1 1701255. aacgaucaL96 1701256. guaUfcAfgucuususc CAGAACGAUCG 1 1 AD- A- 4157 ascsggu(Ahd)CfuUfAfUfuu A- 131 VPusGfsgaua(Tgn)uaa AUACGGUACUUA 4225 901376.1 1701309. aauauccaL96 1701310. auaAfgUfaccgusasu UUUAAUAUCCC 1 1 AD- A- 3 csasgaa(Chd)AfgUfCfCfuu A- 132 VPusCfsugga(Tgn)uaa GACAGAACAGUCC 4226 901356.1 1701269. aauccagaL96 1701270. ggaCfuGfuucugsusc UUAAUCCAGA 1 1 AD- A- 4 csgsaca(Ghd)AfaCfAfGfuc A- 133 VPusGfsauua(Agn)gg AUCGACAGAACAG 4227 901355.1 1701267. cuuaaucaL96 1701268. acugUfuCfugucgsasu UCCUUAAUCC 1 1 AD- A- 5 gscsauu(Uhd)GfuUfUfGfua A- 134 VPusGfsaucu(Tgn)gua AAGCAUUUGUUU 4228 901407.1 1701371. caagaucaL96 1701372. caaAfcAfaaugcsusu GUACAAGAUCC 1 1 AD- A- 6 usasuug(Ghd)UfgUfCfUfuc A- 135 VPusAfsucca(Ggn)ug GUUAUUGGUGUC 4229 901367.1 1701291. acuggauaL96 1701292. aagaCfaCfcaauasasc UUCACUGGAUG 1 1 AD- A- 7 ascsuga(Uhd)AfcAfGfAfac A- 136 VPusAfsucga(Tgn)cgu AGACUGAUACAG 4230 901352.1 1701261. gaucgauaL96 1701262. ucuGfuAfucaguscsu AACGAUCGAUA 1 1 AD- A- 8 asasaga(Chd)UfgAfUfAfca A- 137 VPusAfsucgu(Tgn)cu GGAAAGACUGAU 4231 901348.1 1701253. gaacgauaL96 1701254. guauCfaGfucuuuscsc ACAGAACGAUC 1 1 AD- A- 9 asusaca(Ghd)AfaCfGfAfuc A- 138 VPusCfsugua(Tgn)cga UGAUACAGAACG 4232 901354.1 1701265. gauacagaL96 1701266. ucgUfuCfuguauscsa AUCGAUACAGA 1 1 AD- A- 10 csusgau(Ahd)CfaGfAfAfcg A- 139 VPusUfsaucg(Agn)uc GACUGAUACAGA 4233 901353.1 1701263. aucgauaaL96 1701264. guucUfgUfaucagsusc ACGAUCGAUAC 1 1 AD- A- 11 gsasgaa(Ahd)GfuGfUfUfuu A- 140 VPusCfsguau(Agn)ua AAGAGAAAGUGU 4234 901375.1 1701307. auauacgaL96 1701308. aaacAfcUfuucucsusu UUUAUAUACGG 1 1 AD- A- 12 ascsgaa(Chd)GfuAfCfUfug A- 141 VPusAfscauc(Tgn)gca AAACGAACGUACU 4235 901345.1 1701247. cagauguaL96 1701248. aguAfcGfuucgususu UGCAGAUGUG 1 1 AD- A- 13 csusugg(Ahd)AfuUfGfGfa A- 142 VPusAfsuggc(Ggn)aa CUCUUGGAAUUG 4236 901357.1 1701271. uucgccauaL96 1701272. uccaAfuUfccaagsasg GAUUCGCCAUU 1 1 AD- A- 14 gsgscag(Chd)UfuGfAfGfuu A- 143 VPusUfsucgu(Tgn)uaa GAGGCAGCUUGA 4237 901334.1 1701225. aaacgaaaL96 1701226. cucAfaGfcugccsusc GUUAAACGAAC 1 1 AD- A- 15 gsgsgca(Ghd)AfaUfCfAfuc A- 144 VPusAfscuuc(Ggn)ug GAGGGCAGAAUC 4238 901313.1 1701183. acgaaguaL96 1701184. augaUfuCfugcccsusc AUCACGAAGUG 1 1 AD- A- 16 ususaaa(Chd)GfaAfCfGfua A- 145 VPusCfsugca(Agn)gu AGUUAAACGAAC 4239 901344.1 1701245. cuugcagaL96 1701246. acguUfcGfuuuaascsu GUACUUGCAGA 1 1 AD- A- 17 gsusuau(Uhd)GfgUfGfUfc A- 146 VPusCfscagu(Ggn)aag AUGUUAUUGGUG 4240 901366.1 1701289. uucacuggaL96 1701290. acaCfcAfauaacsasu UCUUCACUGGA 1 1 AD- A- 18 asgscuu(Ghd)AfgUfUfAfaa A- 147 VPusAfscguu(Cgn)gu GCAGCUUGAGUU 4241 901337.1 1701231. cgaacguaL96 1701232. uuaaCfuCfaagcusgsc AAACGAACGUA 1 1 AD- A- 19 gscsagc(Uhd)UfgAfGfUfua A- 148 VPusGfsuucg(Tgn)uu AGGCAGCUUGAG 4242 901335.1 1701227. aacgaacaL96 1701228. aacuCfaAfgcugcscsu UUAAACGAACG 1 1 AD- A- 20 csgsaag(Uhd)GfgUfGfAfag A- 149 VPusCfscaug(Agn)acu CACGAAGUGGUG 4243 901398.1 1701353. uucauggaL96 1701354. ucaCfcAfcuucgsusg AAGUUCAUGGA 1 1 AD- A- 21 csasgaa(Uhd)CfaUfCfAfcg A- 150 VPusAfsccac(Tgn)ucg GGCAGAAUCAUCA 4244 901314.1 1701185. aagugguaL96 1701186. ugaUfgAfuucugscsc CGAAGUGGUG 1 1 AD- A- 22 asasaau(Ahd)GfaCfAfUfug A- 151 VPusAfsgaau(Agn)gc AUAAAAUAGACA 4245 901386.1 1701329. cuauucuaL96 1701330. aaugUfcUfauuuusasu UUGCUAUUCUG 1 1 AD- A- 23 csasgcu(Uhd)GfaGfUfUfaa A- 152 VPusCfsguuc(Ggn)uu GGCAGCUUGAGU 4246 901336.1 1701229. acgaacgaL96 1701230. uaacUfcAfagcugscsc UAAACGAACGU 1 1 AD- A- 24 csgscac(Uhd)GfaAfAfCfuu A- 153 VPusGfsgacg(Agn)aaa GUCGCACUGAAAC 4247 901310.1 1701177. uucguccaL96 1701178. guuUfcAfgugcgsasc UUUUCGUCCA 1 1 AD- A- 25 asgsauu(Ahd)UfgCfGfGfau A- 154 VPusAfsgguu(Tgn)ga GCAGAUUAUGCG 4248 901321.1 1701199. caaaccuaL96 1701200. uccgCfaUfaaucusgsc GAUCAAACCUC 1 1 AD- A- 26 gscsucu(Chd)UfuAfUfUfug A- 155 VPusAfsccgg(Tgn)aca UUGCUCUCUUAUU 4249 901382.1 1701321. uaccgguaL96 1701322. aauAfaGfagagcsasa UGUACCGGUU 1 1 AD- A- 27 usgsaca(Ghd)UfcAfCfUfag A- 156 VPusAfsgaua(Agn)gc GGUGACAGUCACU 4250 901384.1 1701325. cuuaucuaL96 1701326. uaguGfaCfugucascsc AGCUUAUCUU 1 1 AD- A- 28 csusuga(Ghd)UfuAfAfAfcg A- 157 VPusGfsuacg(Tgn)ucg AGCUUGAGUUAA 4251 901339.1 1701235. aacguacaL96 1701236. uuuAfaCfucaagscsu ACGAACGUACU 1 1 AD- A- 29 asgsugc(Uhd)AfaUfGfUfua A- 158 VPusAfscacc(Agn)aua ACAGUGCUAAUG 4252 901363.1 1701283. uugguguaL96 1701284. acaUfuAfgcacusgsu UUAUUGGUGUC 1 1 AD- A- 30 asusccg(Chd)AfgAfCfGfug A- 159 VPusAfscauu(Tgn)aca AGAUCCGCAGACG 4253 901325.1 1701207. uaaauguaL96 1701208. cguCfuGfcggauscsu UGUAAAUGUU 1 1 AD- A- 31 asgsacu(Ghd)AfuAfCfAfga A- 160 VPusCfsgauc(Ggn)uu AAAGACUGAUAC 4254 901350.1 1701257. acgaucgaL96 1701258. cuguAfuCfagucususu AGAACGAUCGA 1 1 AD- A- 32 usgsuua(Uhd)UfgGfUfGfu A- 161 VPusCfsagug(Agn)ag AAUGUUAUUGGU 4255 901365.1 1701287. cuucacugaL96 1701288. acacCfaAfuaacasusu GUCUUCACUGG 1 1 AD- A- 33 gsusgcu(Ghd)GfaAfUfUfu A- 162 VPusUfsgaau(Agn)uc CGGUGCUGGAAU 4256 901306.1 1701169. gauauucaaL96 1701170. aaauUfcCfagcacscsg UUGAUAUUCAU 1 1 AD- A- 34 ususgcu(Ghd)CfuAfAfAfuc A- 163 VPusGfscucg(Ggn)ug UCUUGCUGCUAAA 4257 901361.1 1701279. accgagcaL96 1701280. auuuAfgCfagcaasgsa UCACCGAGCC 1 1 AD- A- 35 csascca(Uhd)GfcAfGfAfuu A- 164 VPusUfsccgc(Agn)uaa AUCACCAUGCAGA 4258 901320.1 1701197. augcggaaL96 1701198. ucuGfcAfuggugsasu UUAUGCGGAU 1 1 AD- A- 36 gsasaag(Chd)AfuUfUfGfuu A- 165 VPusUfsugua(Cgn)aaa GAGAAAGCAUUU 4259 901405.1 1701367. uguacaaaL96 1701368. caaAfuGfcuuucsusc GUUUGUACAAG 1 1 AD- A- 37 gscsuug(Ahd)GfuUfAfAfac A- 166 VPusUfsacgu(Tgn)cgu CAGCUUGAGUUA 4260 901338.1 1701233. gaacguaaL96 1701234. uuaAfcUfcaagcsusg AACGAACGUAC 1 1 AD- A- 38 uscsggu(Ghd)AfcAfGfUfca A- 167 VPusAfsagcu(Agn)gu GAUCGGUGACAG 4261 901383.1 1701323. cuagcuuaL96 1701324. gacuGfuCfaccgasusc UCACUAGCUUA 1 1 AD- A- 39 asgsgca(Ghd)CfuUfGfAfgu A- 168 VPusUfscguu(Tgn)aac CGAGGCAGCUUGA 4262 901333.1 1701223. uaaacgaaL96 1701224. ucaAfgCfugccuscsg GUUAAACGAA 1 1 AD- A- 40 csusgca(Ahd)AfaAfCfAfca A- 169 VPusGfscgag(Tgn)cug UCCUGCAAAAACA 4263 901330.1 1701217. gacucgcaL96 1701218. uguUfuUfugcagsgsa CAGACUCGCG 1 1 AD- A- 41 csusugc(Uhd)GfcUfAfAfau A- 170 VPusCfsucgg(Tgn)gau UUCUUGCUGCUAA 4264 901360.1 1701277. caccgagaL96 1701278. uuaGfcAfgcaagsasa AUCACCGAGC 1 1 AD- A- 42 ususcuu(Ghd)CfuGfCfUfaa A- 171 VPusCfsggug(Agn)uu UUUUCUUGCUGCU 4265 901358.1 1701273. aucaccgaL96 1701274. uagcAfgCfaagaasasa AAAUCACCGA 1 1 AD- A- 43 asasagc(Ahd)UfuUfGfUfuu A- 172 VPusCfsuugu(Agn)ca AGAAAGCAUUUG 4266 901406.1 1701369. guacaagaL96 1701370. aacaAfaUfgcuuuscsu UUUGUACAAGA 1 1 AD- A- 44 uscscgc(Ahd)GfaCfGfUfgu A- 173 VPusAfsacau(Tgn)uac GAUCCGCAGACGU 4267 901326.1 1701209. aaauguuaL96 1701210. acgUfcUfgcggasusc GUAAAUGUUC 1 1 AD- A- 45 csgsgua(Chd)UfuAfUfUfua A- 174 VPusGfsggau(Agn)uu UACGGUACUUAU 4268 901377.1 1701311. auaucccaL96 1701312. aaauAfaGfuaccgsusa UUAAUAUCCCU 1 1 AD- A- 46 gsascug(Ahd)UfaCfAfGfaa A- 175 VPusUfscgau(Cgn)gu AAGACUGAUACA 4269 901351.1 1701259. cgaucgaaL96 1701260. ucugUfaUfcagucsusu GAACGAUCGAU 1 1 AD- A- 47 asasaac(Ahd)CfaGfAfCfuc A- 176 VPusGfscaac(Ggn)cga CAAAAACACAGAC 4270 901415.1 1701387. gcguugcaL96 1701388. gucUfgUfguuuususg UCGCGUUGCA 1 1 AD- A- 48 gsasguu(Ahd)AfaCfGfAfac A- 177 VPusCfsaagu(Agn)cg UUGAGUUAAACG 4271 901342.1 1701241. guacuugaL96 1701242. uucgUfuUfaacucsasa AACGUACUUGC 1 1 AD- A- 49 uscsacu(Ghd)GfaUfGfUfau A- 178 VPusCfsaguc(Agn)aau CUUCACUGGAUGU 4272 901420.1 1701397. uugacugaL96 1701398. acaUfcCfagugasasg AUUUGACUGC 1 1 AD- A- 50 cscsucc(Ghd)AfaAfCfCfau A- 179 VPusAfsaagu(Tgn)cau GGCCUCCGAAACC 4273 901312.1 1701181. gaacuuuaL96 1701182. gguUfuCfggaggscsc AUGAACUUUC 1 1 AD- A- 51 ususgag(Uhd)UfaAfAfCfga A- 180 VPusAfsguac(Ggn)uu GCUUGAGUUAAA 4274 901340.1 1701237. acguacuaL96 1701238. cguuUfaAfcucaasgsc CGAACGUACUU 1 1 AD- A- 52 usgscua(Chd)UfgUfUfUfau A- 181 VPusAfsuuac(Ggn)ga GGUGCUACUGUU 4275 901392.1 1701341. ccguaauaL96 1701342. uaaaCfaGfuagcascsc UAUCCGUAAUA 1 1 AD- A- 53 cscsgca(Ghd)AfcGfUfGfua A- 182 VPusGfsaaca(Tgn)uua AUCCGCAGACGUG 4276 901327.1 1701211. aauguucaL96 1701212. cacGfuCfugcggsasu UAAAUGUUCC 1 1 AD- A- 54 csgscag(Ahd)CfgUfGfUfaa A- 183 VPusGfsgaac(Agn)uu UCCGCAGACGUGU 4277 901328.1 1701213. auguuccaL96 1701214. uacaCfgUfcugcgsgsa AAAUGUUCCU 1 1 AD- A- 55 asgsaga(Ahd)GfaGfAfCfac A- 184 VPusCfsaaca(Agn)ugu GAAGAGAAGAGA 4278 901370.1 1701297. auuguugaL96 1701298. gucUfcUfucucususc CACAUUGUUGG 1 1 AD- A- 56 ascsagc(Ahd)CfaAfCfAfaa A- 185 VPusAfsuuca(Cgn)au CUACAGCACAACA 4279 901399.1 1701355. ugugaauaL96 1701356. uuguUfgUfgcugusasg AAUGUGAAUG 1 1 AD- A- 57 uscsuug(Chd)UfgCfUfAfaa A- 186 VPusUfscggu(Ggn)au UUUCUUGCUGCUA 4280 901359.1 1701275. ucaccgaaL96 1701276. uuagCfaGfcaagasasa AAUCACCGAG 1 1 AD- A- 58 ascsacc(Ahd)UfuGfAfAfac A- 187 VPusAfscuag(Tgn)gg UCACACCAUUGAA 4281 901373.1 1701303. cacuaguaL96 1701304. uuucAfaUfggugusgsa ACCACUAGUU 1 1 AD- A- 59 gsasggc(Ahd)GfcUfUfGfag A- 188 VPusCfsguuu(Agn)ac GCGAGGCAGCUUG 4282 901332.1 1701221. uuaaacgaL96 1701222. ucaaGfcUfgccucsgsc AGUUAAACGA 1 1 AD- A- 60 gscsacu(Ghd)AfaAfCfUfuu A- 189 VPusUfsggac(Ggn)aaa UCGCACUGAAACU 4283 901311.1 1701179. ucguccaaL96 1701180. aguUfuCfagugcsgsa UUUCGUCCAA 1 1 AD- A- 61 gsusuuu(Ahd)UfaUfAfCfg A- 190 VPusAfsuaag(Tgn)acc GUGUUUUAUAUA 4284 901423.1 1701403. guacuuauaL96 1701404. guaUfaUfaaaacsasc CGGUACUUAUU 1 1 AD- A- 62 csascca(Uhd)UfgAfAfAfcc A- 191 VPusAfsacua(Ggn)ug CACACCAUUGAAA 4285 901374.1 1701305. acuaguuaL96 1701306. guuuCfaAfuggugsusg CCACUAGUUC 1 1 AD- A- 63 asuscac(Chd)AfuGfCfAfga A- 192 VPusCfsgcau(Agn)auc ACAUCACCAUGCA 4286 901319.1 1701195. uuaugcgaL96 1701196. ugcAfuGfgugausgsu GAUUAUGCGG 1 1 AD- A- 64 usgsagu(Uhd)AfaAfCfGfaa A- 193 VPusAfsagua(Cgn)gu CUUGAGUUAAAC 4287 901341.1 1701239. cguacuuaL96 1701240. ucguUfuAfacucasasg GAACGUACUUG 1 1 AD- A- 65 gsasaag(Uhd)GfuUfUfUfau A- 194 VPusAfsccgu(Agn)ua GAGAAAGUGUUU 4288 901422.1 1701401. auacgguaL96 1701402. uaaaAfcAfcuuucsusc UAUAUACGGUA 1 1 AD- A- 66 ascsagu(Chd)AfcUfAfGfcu A- 195 VPusCfsaaga(Tgn)aag UGACAGUCACUAG 4289 901385.1 1701327. uaucuugaL96 1701328. cuaGfuGfacuguscsa CUUAUCUUGA 1 1 AD- A- 67 gsusgcu(Ahd)CfuGfUfUfua A- 196 VPusUfsuacg(Ggn)au UGGUGCUACUGU 4290 901391.1 1701339. uccguaaaL96 1701340. aaacAfgUfagcacscsa UUAUCCGUAAU 1 1 AD- A- 68 cscsugc(Ahd)AfaAfAfCfac A- 197 VPusCfsgagu(Cgn)ug UUCCUGCAAAAAC 4291 901329.1 1701215. agacucgaL96 1701216. uguuUfuUfgcaggsasa ACAGACUCGC 1 1 AD- A- 69 csasaaa(Ahd)CfaCfAfGfac A- 198 VPusAfsacgc(Ggn)ag UGCAAAAACACAG 4292 901331.1 1701219. ucgcguuaL96 1701220. ucugUfgUfuuuugscsa ACUCGCGUUG 1 1 AD- A- 70 usgscug(Uhd)GfgAfCfUfu A- 199 VPusCfsccaa(Cgn)uca ACUGCUGUGGACU 4293 901368.1 1701293. gaguugggaL96 1701294. aguCfcAfcagcasgsu UGAGUUGGGA 1 1 AD- A- 71 gsusgcu(Ahd)AfuGfUfUfa A- 200 VPusGfsacac(Cgn)aau CAGUGCUAAUGU 4294 901364.1 1701285. uuggugucaL96 1701286. aacAfuUfagcacsusg UAUUGGUGUCU 1 1 AD- A- 72 usgsgug(Chd)UfaCfUfGfuu A- 201 VPusAfscgga(Tgn)aaa AUUGGUGCUACU 4295 901389.1 1701335. uauccguaL96 1701336. cagUfaGfcaccasasu GUUUAUCCGUA 1 1 AD- A- 73 asgsaaa(Ghd)UfgUfUfUfua A- 202 VPusCfscgua(Tgn)aua AGAGAAAGUGUU 4296 901421.1 1701399. uauacggaL96 1701400. aaaCfaCfuuucuscsu UUAUAUACGGU 1 1 AD- A- 74 asascua(Uhd)UfuAfUfGfag A- 203 VPusGfsauac(Agn)uc UCAACUAUUUAU 4297 901380.1 1701317. auguaucaL96 1701318. ucauAfaAfuaguusgsa GAGAUGUAUCU 1 1 AD- A- 75 asgsuua(Ahd)AfcGfAfAfcg A- 204 VPusGfscaag(Tgn)acg UGAGUUAAACGA 4298 901343.1 1701243. uacuugcaL96 1701244. uucGfuUfuaacuscsa ACGUACUUGCA 1 1 AD- A- 76 csasucu(Uhd)CfaAfGfCfca A- 205 VPusCfsacag(Ggn)aug UACAUCUUCAAGC 4299 901317.1 1701191. uccugugaL96 1701192. gcuUfgAfagaugsusa CAUCCUGUGU 1 1 AD- A- 77 ususuuu(Uhd)UfuCfAfGfu A- 206 VPusCfscaag(Agn)aua GGUUUUUUUUCA 4300 901424.1 1701405. auucuuggaL96 1701406. cugAfaAfaaaaascsc GUAUUCUUGGU 1 1 AD- A- 78 ususauc(Chd)GfuAfAfUfaa A- 207 VPusCfsccac(Agn)auu GUUUAUCCGUAA 4301 901431.1 1701419. uugugggaL96 1701420. auuAfcGfgauaasasc UAAUUGUGGGG 1 1 AD- A- 79 gsgsuac(Uhd)UfaUfUfUfaa A- 208 VPusAfsggga(Tgn)au ACGGUACUUAUU 4302 901378.1 1701313. uaucccuaL96 1701314. uaaaUfaAfguaccsgsu UAAUAUCCCUU 1 1 AD- A- 80 csascgu(Chd)UfuUfGfUfcu A- 209 VPusGfscacu(Agn)ga AUCACGUCUUUGU 4303 901434.1 1701425. cuagugcaL96 1701426. gacaAfaGfacgugsasu CUCUAGUGCA 1 1 AD- A- 81 usgscaa(Ahd)AfaCfAfCfag A- 210 VPusCfsgcga(Ggn)uc CCUGCAAAAACAC 4304 901412.1 1701381. acucgcgaL96 1701382. ugugUfuUfuugcasgsg AGACUCGCGU 1 1 AD- A- 82 ascsuau(Uhd)UfaUfGfAfga A- 211 VPusAfsgaua(Cgn)auc CAACUAUUUAUG 4305 901426.1 1701409. uguaucuaL96 1701410. ucaUfaAfauagususg AGAUGUAUCUU 1 1 AD- A- 83 asgsggg(Chd)AfaAfAfAfcg A- 212 VPusGfscgcu(Tgn)ucg AAAGGGGCAAAA 4306 901322.1 1701201. aaagcgcaL96 1701202. uuuUfuGfccccususu ACGAAAGCGCA 1 1 AD- A- 84 ususgcu(Chd)UfcUfUfAfuu A- 213 VPusCfsggua(Cgn)aaa UCUUGCUCUCUUA 4307 901381.1 1701319. uguaccgaL96 1701320. uaaGfaGfagcaasgsa UUUGUACCGG 1 1 AD- A- 85 asusuug(Uhd)UfuGfUfAfca A- 214 VPusCfsggau(Cgn)uu GCAUUUGUUUGU 4308 901324.1 1701205. agauccgaL96 1701206. guacAfaAfcaaausgsc ACAAGAUCCGC 1 1 AD- A- 86 ususgca(Ghd)AfuGfUfGfac A- 215 VPusUfscggc(Tgn)ug ACUUGCAGAUGU 4309 901347.1 1701251. aagccgaaL96 1701252. ucacAfuCfugcaasgsu GACAAGCCGAG 1 1 AD- A- 87 csasacu(Ahd)UfuUfAfUfga A- 216 VPusAfsuaca(Tgn)cuc UUCAACUAUUUA 4310 901379.1 1701315. gauguauaL96 1701316. auaAfaUfaguugsasa UGAGAUGUAUC 1 1 AD- A- 88 asasuuc(Uhd)AfcAfUfAfcu A- 217 VPusGfsagau(Tgn)uag AGAAUUCUACAU 4311 901428.1 1701413. aaaucucaL96 1701414. uauGfuAfgaauuscsu ACUAAAUCUCU 1 1 AD- A- 89 asusguc(Chd)UfcAfCfAfcc A- 218 VPusUfsuuca(Agn)ug CUAUGUCCUCACA 4312 901371.1 1701299. auugaaaaL96 1701300. guguGfaGfgacausasg CCAUUGAAAC 1 1 AD- A- 90 ususguu(Uhd)GfuAfCfAfa A- 219 VPusUfsgcgg(Agn)uc AUUUGUUUGUAC 4313 901408.1 1701373. gauccgcaaL96 1701374. uuguAfcAfaacaasasu AAGAUCCGCAG 1 1 AD- A- 91 usasauc(Chd)AfgAfAfAfcc A- 220 VPusCfsauuu(Cgn)ag CUUAAUCCAGAAA 4314 901417.1 1701391. ugaaaugaL96 1701392. guuuCfuGfgauuasasg CCUGAAAUGA 1 1 AD- A- 92 gsasaaa(Ahd)AfaAfUfCfag A- 221 VPusCfscucg(Agn)acu AAGAAAAAAAAU 4315 901400.1 1701357. uucgaggaL96 1701358. gauUfuUfuuuucsusu CAGUUCGAGGA 1 1 AD- A- 93 gsgsgca(Ahd)AfaAfCfGfaa A- 222 VPusUfsugcg(Cgn)uu AGGGGCAAAAAC 4316 901323.1 1701203. agcgcaaaL96 1701204. ucguUfuUfugcccscsu GAAAGCGCAAG 1 1 AD- A- 94 usgsaag(Uhd)UfcAfUfGfga A- 223 VPusAfsuaga(Cgn)auc GGUGAAGUUCAU 4317 901316.1 1701189. ugucuauaL96 1701190. cauGfaAfcuucascsc GGAUGUCUAUC 1 1 AD- A- 95 csascga(Ahd)GfuGfGfUfga A- 224 VPusAfsugaa(Cgn)uu AUCACGAAGUGG 4318 901315.1 1701187. aguucauaL96 1701188. caccAfcUfucgugsasu UGAAGUUCAUG 1 1 AD- A- 96 ascsguc(Uhd)UfuGfUfCfuc A- 225 VPusUfsgcac(Tgn)aga UCACGUCUUUGUC 4319 901395.1 1701347. uagugcaaL96 1701348. gacAfaAfgacgusgsa UCUAGUGCAG 1 1 AD- A- 97 asascau(Chd)AfcCfAfUfgc A- 226 VPusAfsuaau(Cgn)ug CCAACAUCACCAU 4320 901318.1 1701193. agauuauaL96 1701194. caugGfuGfauguusgsg GCAGAUUAUG 1 1 AD- A- 98 gsgsugc(Uhd)AfcUfGfUfu A- 227 VPusUfsacgg(Agn)ua UUGGUGCUACUG 4321 901390.1 1701337. uauccguaaL96 1701338. aacaGfuAfgcaccsasa UUUAUCCGUAA 1 1 AD- A- 99 asasaua(Ghd)AfcAfUfUfgc A- 228 VPusCfsagaa(Tgn)agc UAAAAUAGACAU 4322 901387.1 1701331. uauucugaL96 1701332. aauGfuCfuauuususa UGCUAUUCUGU 1 1 AD- A- 100 ususccc(Chd)AfaAfUfCfac A- 229 VPusAfsucca(Cgn)agu ACUUCCCCAAAUC 4323 901307.1 1701171. uguggauaL96 1701172. gauUfuGfgggaasgsu ACUGUGGAUU 1 1 AD- A- 101 gsasucc(Ghd)CfaGfAfCfgu A- 230 VPusCfsauuu(Agn)cac AAGAUCCGCAGAC 4324 901410.1 1701377. guaaaugaL96 1701378. gucUfgCfggaucsusu GUGUAAAUGU 1 1 AD- A- 102 ususaac(Ahd)UfcAfCfGfuc A- 231 VPusAfsgaca(Agn)aga UAUUAACAUCACG 4325 901433.1 1701423. uuugucuaL96 1701424. cguGfaUfguuaasusa UCUUUGUCUC 1 1 AD- A- 103 asasagu(Ghd)AfgUfGfAfcc A- 232 VPusAfsaaag(Cgn)agg GCAAAGUGAGUG 4326 901308.1 1701173. ugcuuuuaL96 1701174. ucaCfuCfacuuusgsc ACCUGCUUUUG 1 1 AD- A- 104 asasaaa(Chd)AfcAfGfAfcu A- 233 VPusCfsaacg(Cgn)gag GCAAAAACACAGA 4327 901414.1 1701385. cgcguugaL96 1701386. ucuGfuGfuuuuusgsc CUCGCGUUGC 1 1 AD- A- 105 csgsucg(Chd)AfcUfGfAfaa A- 234 VPusCfsgaaa(Agn)gu GGCGUCGCACUGA 4328 901309.1 1701175. cuuuucgaL96 1701176. uucaGfuGfcgacgscsc AACUUUUCGU 1 1 AD- A- 106 asascag(Uhd)GfcUfAfAfug A- 235 VPusCfscaau(Agn)aca UUAACAGUGCUA 4329 901362.1 1701281. uuauuggaL96 1701282. uuaGfcAfcuguusasa AUGUUAUUGGU 1 1 AD- A- 107 ususcgu(Chd)CfaAfCfUfuc A- 236 VPusCfsagcc(Cgn)aga UUUUCGUCCAACU 4330 901397.1 1701351. ugggcugaL96 1701352. aguUfgGfacgaasasa UCUGGGCUGU 1 1 AD- A- 108 asusugg(Uhd)GfuCfUfUfca A- 237 VPusCfsaucc(Agn)gu UUAUUGGUGUCU 4331 901419.1 1701395. cuggaugaL96 1701396. gaagAfcAfccaausasa UCACUGGAUGU 1 1 AD- A- 109 gscsaaa(Ahd)AfcAfCfAfga A- 238 VPusAfscgcg(Agn)gu CUGCAAAAACACA 4332 901413.1 1701383. cucgcguaL96 1701384. cuguGfuUfuuugcsasg GACUCGCGUU 1 1 AD- A- 110 asasaaa(Uhd)CfaGfUfUfcg A- 239 VPusCfsuuuc(Cgn)uc AAAAAAAUCAGU 4333 901401.1 1701359. aggaaagaL96 1701360. gaacUfgAfuuuuususu UCGAGGAAAGG 1 1 AD- A- ill csasgac(Ghd)UfgUfAfAfau A- 240 VPusCfsagga(Agn)cau CGCAGACGUGUAA 4334 901411.1 1701379. guuccugaL96 1701380. uuaCfaCfgucugscsg AUGUUCCUGC 1 1 AD- A- 112 usgsucc(Uhd)CfaCfAfCfca A- 241 VPusGfsuuuc(Agn)au UAUGUCCUCACAC 4335 901372.1 1701301. uugaaacaL96 1701302. ggugUfgAfggacasusa CAUUGAAACC 1 1 AD- A- 113 ususuuu(Uhd)UfcAfGfUfa A- 242 VPusAfsccaa(Ggn)aau GUUUUUUUUCAG 4336 901425.1 1701407. uucuugguaL96 1701408. acuGfaAfaaaaasasc UAUUCUUGGUU 1 1 AD- A- 114 asgsauc(Chd)GfcAfGfAfcg A- 243 VPusAfsuuua(Cgn)ac CAAGAUCCGCAGA 4337 901409.1 1701375. uguaaauaL96 1701376. gucuGfcGfgaucususg CGUGUAAAUG 1 1 AD- A- 115 cscscuc(Uhd)UfgGfAfAfuu A- 244 VPusCfsgaau(Cgn)caa GUCCCUCUUGGAA 4338 901418.1 1701393. ggauucgaL96 1701394. uucCfaAfgagggsasc UUGGAUUCGC 1 1 AD- A- 116 gsasuau(Uhd)AfaCfAfUfca A- 245 VPusAfsaaga(Cgn)gu AAGAUAUUAACA 4339 901393.1 1701343. cgucuuuaL96 1701344. gaugUfuAfauaucsusu UCACGUCUUUG 1 1 AD- A- 117 ususggu(Ghd)CfuAfCfUfg A- 246 VPusCfsggau(Agn)aac UAUUGGUGCUAC 4340 901388.1 1701333. uuuauccgaL96 1701334. aguAfgCfaccaasusa UGUUUAUCCGU 1 1 AD- A- 118 asasggg(Ghd)CfaAfAfAfac A- 247 VPusCfsgcuu(Tgn)cgu GAAAGGGGCAAA 4341 901404.1 1701365. gaaagcgaL96 1701366. uuuUfgCfcccuususc AACGAAAGCGC 1 1 AD- A- 119 csusugc(Ahd)GfaUfGfUfga A- 248 VPusCfsggcu(Tgn)guc UACUUGCAGAUG 4342 901346.1 1701249. caagccgaL96 1701250. acaUfcUfgcaagsusa UGACAAGCCGA 1 1 AD- A- 120 asasauc(Ahd)GfuUfCfGfag A- 249 VPusCfsccuu(Tgn)ccu AAAAAUCAGUUC 4343 901403.1 1701363. gaaagggaL96 1701364. cgaAfcUfgauuususu GAGGAAAGGGA 1 1 AD- A- 121 ascsuuu(Uhd)CfgUfCfCfaa A- 250 VPusCfscaga(Agn)gu AAACUUUUCGUCC 4344 901396.1 1701349. cuucuggaL96 1701350. uggaCfgAfaaagususu AACUUCUGGG 1 1 AD- A- 122 asusuaa(Chd)AfuCfAfCfgu A- 251 VPusGfsacaa(Agn)gac AUAUUAACAUCAC 4345 901432.1 1701421. cuuugucaL96 1701422. gugAfuGfuuaausasu GUCUUUGUCU 1 1 AD- A- 123 csgsucu(Uhd)UfgUfCfUfcu A- 252 VPusCfsugca(Cgn)uag CACGUCUUUGUCU 4346 901435.1 1701427. agugcagaL96 1701428. agaCfaAfagacgsusg CUAGUGCAGU 1 1 AD- A- 124 asascac(Ahd)GfaCfUfCfgc A- 253 VPusUfsugca(Agn)cg AAAACACAGACUC 4347 901416.1 1701389. guugcaaaL96 1701390. cgagUfcUfguguususu GCGUUGCAAG 1 1 AD- A- 125 asusauu(Ahd)AfcAfUfCfac A- 254 VPusCfsaaag(Agn)cgu AGAUAUUAACAU 4348 901394.1 1701345. gucuuugaL96 1701346. gauGfuUfaauauscsu CACGUCUUUGU 1 1 AD- A- 126 gsusuua(Uhd)CfcGfUfAfau A- 255 VPusCfsacaa(Tgn)uau CUGUUUAUCCGUA 4349 901429.1 1701415. aauugugaL96 1701416. uacGfgAfuaaacsasg AUAAUUGUGG 1 1 AD- A- 127 asasaau(Chd)AfgUfUfCfga A- 256 VPusCfscuuu(Cgn)cuc AAAAAAUCAGUU 4350 901402.1 1701361. ggaaaggaL96 1701362. gaaCfuGfauuuususu CGAGGAAAGGG 1 1 AD- A- 128 ususuau(Chd)CfgUfAfAfua A- 257 VPusCfscaca(Agn)uua UGUUUAUCCGUA 4351 901430.1 1701417. auuguggaL96 1701418. uuaCfgGfauaaascsa AUAAUUGUGGG 1 1 AD- A- 129 asgsgac(Ahd)UfuGfCfUfgu A- 258 VPusCfscaaa(Ggn)cac UCAGGACAUUGCU 4352 901369.1 1701295. gcuuuggaL96 1701296. agcAfaUfguccusgsa GUGCUUUGGG 1 1

TABLE 2B Exemplary Human VEGF-A siRNA Unmodified Single Strands and Duplex Sequences Sense SEQ ID mRNA Antisense SEQ ID mRNA Duplex Oligo NO: Target Oligo NO: Target Name Name (Sense) Sense Sequence Range Name (Antisense) Antisense Sequence Range AD- A- 259 AAGACUGAUACAGAA 1796- A- 388 UGAUCGTUCUGUAUCAGU 1794- 901349. 1701255. CGAUCA 1816 1701256. CUUUC 1816 1 1 1 AD- A- 260 ACGGUACUUAUUUAA 2961- A- 389 UGGAUATUAAAUAAGUAC 2959- 901376. 1701309. UAUCCA 2981 1701310. CGUAU 2981 1 1 1 AD- A- 261 CAGAACAGUCCUUAA 1858- A- 390 UCUGGATUAAGGACUGUU 1856- 901356. 1701269. UCCAGA 1878 1701270. CUGUC 1878 1 1 1 AD- A- 262 CGACAGAACAGUCCU 1855- A- 391 UGAUUAAGGACUGUUCU 1853- 901355. 1701267. UAAUCA 1875 1701268. GUCGAU 1875 1 1 1 AD- A- 263 GCAUUUGUUUGUACA 1614- A- 392 UGAUCUTGUACAAACAAA 1612- 901407. 1701371. AGAUCA 1634 1701372. UGCUU 1634 1 1 1 AD- A- 264 UAUUGGUGUCUUCAC 2192- A- 393 UAUCCAGUGAAGACACCA 2190- 901367. 1701291. UGGAUA 2212 1701292. AUAAC 2212 1 1 1 AD- A- 265 ACUGAUACAGAACGA 1799- A- 394 UAUCGATCGUUCUGUAUC 1797- 901352. 1701261. UCGAUA 1819 1701262. AGUCU 1819 1 1 1 AD- A- 266 AAAGACUGAUACAGA 1795- A- 395 UAUCGUTCUGUAUCAGUC 1793- 901348. 1701253. ACGAUA 1815 1701254. UUUCC 1815 1 1 1 AD- A- 267 AUACAGAACGAUCGA 1803- A- 396 UCUGUATCGAUCGUUCUG 1801- 901354. 1701265. UACAGA 1823 1701266. UAUCA 1823 1 1 1 AD- A- 268 CUGAUACAGAACGAU 1800- A- 397 UUAUCGAUCGUUCUGUAU 1798- 901353. 1701263. CGAUAA 1820 1701264. CAGUC 1820 1 1 1 AD- A- 269 GAGAAAGUGUUUUA 2944- A- 398 UCGUAUAUAAAACACUUU 2942- 901375. 1701307. UAUACGA 2964 1701308. CUCUU 2964 1 1 1 AD- A- 270 ACGAACGUACUUGCA 1700- A- 399 UACAUCTGCAAGUACGUU 1698- 901345. 1701247. GAUGUA 1720 1701248. CGUUU 1720 1 1 1 AD- A- 271 CUUGGAAUUGGAUUC 1982- A- 400 UAUGGCGAAUCCAAUUCC 1980- 901357. 1701271. GCCAUA 2002 1701272. AAGAG 2002 1 1 1 AD- A- 272 GGCAGCUUGAGUUAA 1685- A- 401 UUUCGUTUAACUCAAGCU 1683- 901334. 1701225. ACGAAA 1705 1701226. GCCUC 1705 1 1 1 AD- A- 273 GGGCAGAAUCAUCAC 1138- A- 402 UACUUCGUGAUGAUUCUG 1136- 901313. 1701183. GAAGUA 1158 1701184. CCCUC 1158 1 1 1 AD- A- 274 UUAAACGAACGUACU 1696- A- 403 UCUGCAAGUACGUUCGUU 1694- 901344. 1701245. UGCAGA 1716 1701246. UAACU 1716 1 1 1 AD- A- 275 GUUAUUGGUGUCUUC 2190- A- 404 UCCAGUGAAGACACCAAU 2188- 901366. 1701289. ACUGGA 2210 1701290. AACAU 2210 1 1 1 AD- A- 276 AGCUUGAGUUAAACG 1688- A- 405 UACGUUCGUUUAACUCAA 1686- 901337. 1701231. AACGUA 1708 1701232. GCUGC 1708 1 1 1 AD- A- 277 GCAGCUUGAGUUAAA 1686- A- 406 UGUUCGTUUAACUCAAGC 1684- 901335. 1701227. CGAACA 1706 1701228. UGCCU 1706 1 1 1 AD- A- 278 CGAAGUGGUGAAGUU 1152- A- 407 UCCAUGAACUUCACCACU 1150- 901398. 1701353. CAUGGA 1172 1701354. UCGUG 1172 1 1 1 AD- A- 279 CAGAAUCAUCACGAA 1141- A- 408 UACCACTUCGUGAUGAUU 1139- 901314. 1701185. GUGGUA 1161 1701186. CUGCC 1161 1 1 1 AD- A- 280 AAAAUAGACAUUGCU 3361- A- 409 UAGAAUAGCAAUGUCUA 3359- 901386. 1701329. AUUCUA 3381 1701330. UUUUAU 3381 1 1 1 AD- A- 281 CAGCUUGAGUUAAAC 1687- A- 410 UCGUUCGUUUAACUCAAG 1685- 901336. 1701229. GAACGA 1707 1701230. CUGCC 1707 1 1 1 AD- A- 282 CGCACUGAAACUUUU  648- A- 411 UGGACGAAAAGUUUCAG  646- 901310. 1701177. CGUCCA  668 1701178. UGCGAC  668 1 1 1 AD- A- 283 AGAUUAUGCGGAUCA 1352- A- 412 UAGGUUTGAUCCGCAUAA 1350- 901321. 1701199. AACCUA 1372 1701200. UCUGC 1372 1 1 1 AD- A- 284 GCUCUCUUAUUUGUA 3096- A- 413 UACCGGTACAAAUAAGAG 3094- 901382. 1701321. CCGGUA 3116 1701322. AGCAA 3116 1 1 1 AD- A- 285 UGACAGUCACUAGCU 3162- A- 414 UAGAUAAGCUAGUGACU 3160- 901384. 1701325. UAUCUA 3182 1701326. GUCACC 3182 1 1 1 AD- A- 286 CUUGAGUUAAACGAA 1690- A- 415 UGUACGTUCGUUUAACUC 1688- 901339. 1701235. CGUACA 1710 1701236. AAGCU 1710 1 1 1 AD- A- 287 AGUGCUAAUGUUAUU 2181- A- 416 UACACCAAUAACAUUAGC 2179- 901363. 1701283. GGUGUA 2201 1701284. ACUGU 2201 1 1 1 AD- A- 288 AUCCGCAGACGUGUA 1631- A- 417 UACAUUTACACGUCUGCG 1629- 901325. 1701207. AAUGUA 1651 1701208. GAUCU 1651 1 1 1 AD- A- 289 AGACUGAUACAGAAC 1797- A- 418 UCGAUCGUUCUGUAUCAG 1795- 901350. 1701257. GAUCGA 1817 1701258. UCUUU 1817 1 1 1 AD- A- 290 UGUUAUUGGUGUCUU 2189- A- 419 UCAGUGAAGACACCAAUA 2187- 901365. 1701287. CACUGA 2209 1701288. ACAUU 2209 1 1 1 AD- A- 291 GUGCUGGAAUUUGAU  125- A- 420 UUGAAUAUCAAAUUCCAG  123- 901306. 1701169. AUUCAA  145 1701170. CACCG  145 1 1 1 AD- A- 292 UUGCUGCUAAAUCAC 2012- A- 421 UGCUCGGUGAUUUAGCAG 2010- 901361. 1701279. CGAGCA 2032 1701280. CAAGA 2032 1 1 1 AD- A- 293 CACCAUGCAGAUUAU 1344- A- 422 UUCCGCAUAAUCUGCAUG 1342- 901320. 1701197. GCGGAA 1364 1701198. GUGAU 1364 1 1 1 AD- A- 294 GAAAGCAUUUGUUUG 1610- A- 423 UUUGUACAAACAAAUGCU 1608- 901405. 1701367. UACAAA 1630 1701368. UUCUC 1630 1 1 1 AD- A- 295 GCUUGAGUUAAACGA 1689- A- 424 UUACGUTCGUUUAACUCA 1687- 901338. 1701233. ACGUAA 1709 1701234. AGCUG 1709 1 1 1 AD- A- 296 UCGGUGACAGUCACU 3158- A- 425 UAAGCUAGUGACUGUCAC 3156- 901383. 1701323. AGCUUA 3178 1701324. CGAUC 3178 1 1 1 AD- A- 297 AGGCAGCUUGAGUUA 1684- A- 426 UUCGUUTAACUCAAGCUG 1682- 901333. 1701223. AACGAA 1704 1701224. CCUCG 1704 1 1 1 AD- A- 298 CUGCAAAAACACAGA 1653- A- 427 UGCGAGTCUGUGUUUUUG 1651- 901330. 1701217. CUCGCA 1673 1701218. CAGGA 1673 1 1 1 AD- A- 299 CUUGCUGCUAAAUCA 2011- A- 428 UCUCGGTGAUUUAGCAGC 2009- 901360. 1701277. CCGAGA 2031 1701278. AAGAA 2031 1 1 1 AD- A- 300 UUCUUGCUGCUAAAU 2009- A- 429 UCGGUGAUUUAGCAGCAA 2007- 901358. 1701273. CACCGA 2029 1701274. GAAAA 2029 1 1 1 AD- A- 301 AAAGCAUUUGUUUGU 1611- A- 430 UCUUGUACAAACAAAUGC 1609- 901406. 1701369. ACAAGA 1631 1701370. uuucu 1631 1 1 1 AD- A- 302 UCCGCAGACGUGUAA 1632- A- 431 UAACAUTUACACGUCUGC 1630- 901326. 1701209. AUGUUA 1652 1701210. GGAUC 1652 1 1 1 AD- A- 303 CGGUACUUAUUUAAU 2962- A- 432 UGGGAUAUUAAAUAAGU 2960- 901377. 1701311. AUCCCA 2982 1701312. ACCGUA 2982 1 1 1 AD- A- 304 GACUGAUACAGAACG 1798- A- 433 UUCGAUCGUUCUGUAUCA 1796- 901351. 1701259. AUCGAA 1818 1701260. GUCUU 1818 1 1 1 AD- A- 305 AAAACACAGACUCGC 1658- A- 434 UGCAACGCGAGUCUGUGU 1656- 901415. 1701387. GUUGCA 1678 1701388. UUUUG 1678 1 1 1 AD- A- 306 GAGUUAAACGAACGU 1693- A- 435 UCAAGUACGUUCGUUUAA 1691- 901342. 1701241. ACUUGA 1713 1701242. CUCAA 1713 1 1 1 AD- A- 307 UCACUGGAUGUAUUU 2203- A- 436 UCAGUCAAAUACAUCCAG 2201- 901420. 1701397. GACUGA 2223 1701398. UGAAG 2223 1 1 1 AD- A- 308 CCUCCGAAACCAUGA 1028- A- 437 UAAAGUTCAUGGUUUCGG 1026- 901312. 1701181. ACUUUA 1048 1701182. AGGCC 1048 1 1 1 AD- A- 309 UUGAGUUAAACGAAC 1691- A- 438 UAGUACGUUCGUUUAACU 1689- 901340. 1701237. GUACUA 1711 1701238. CAAGC 1711 1 1 1 AD- A- 310 UGCUACUGUUUAUCC 3482- A- 439 UAUUACGGAUAAACAGU 3480- 901392. 1701341. GUAAUA 3502 1701342. AGCACC 3502 1 1 1 AD- A- 311 CCGCAGACGUGUAAA 1633- A- 440 UGAACATUUACACGUCUG 1631- 901327. 1701211. UGUUCA 1653 1701212. CGGAU 1653 1 1 1 AD- A- 312 CGCAGACGUGUAAAU 1634- A- 441 UGGAACAUUUACACGUCU 1632- 901328. 1701213. GUUCCA 1654 1701214. GCGGA 1654 1 1 1 AD- A- 313 AGAGAAGAGACACAU 2673- A- 442 UCAACAAUGUGUCUCUUC 2671- 901370. 1701297. UGUUGA 2693 1701298. UCUUC 2693 1 1 1 AD- A- 314 ACAGCACAACAAAUG 1407- A- 443 UAUUCACAUUUGUUGUGC 1405- 901399. 1701355. UGAAUA 1427 1701356. UGUAG 1427 1 1 1 AD- A- 315 UCUUGCUGCUAAAUC 2010- A- 444 UUCGGUGAUUUAGCAGCA 2008- 901359. 1701275. ACCGAA 2030 1701276. AGAAA 2030 1 1 1 AD- A- 316 ACACCAUUGAAACCA 2790- A- 445 UACUAGTGGUUUCAAUGG 2788- 901373. 1701303. CUAGUA 2810 1701304. UGUGA 2810 1 1 1 AD- A- 317 GAGGCAGCUUGAGUU 1683- A- 446 UCGUUUAACUCAAGCUGC 1681- 901332. 1701221. AAACGA 1703 1701222. CUCGC 1703 1 1 1 AD- A- 318 GCACUGAAACUUUUC  649- A- 447 UUGGACGAAAAGUUUCA  647- 901311. 1701179. GUCCAA  669 1701180. GUGCGA  669 1 1 1 AD- A- 319 GUUUUAUAUACGGUA 2952- A- 448 UAUAAGTACCGUAUAUAA 2950- 901423. 1701403. CUUAUA 2972 1701404. AACAC 2972 1 1 1 AD- A- 320 CACCAUUGAAACCAC 2791- A- 449 UAACUAGUGGUUUCAAU 2789- 901374. 1701305. UAGUUA 2811 1701306. GGUGUG 2811 1 1 1 AD- A- 321 AUCACCAUGCAGAUU 1342- A- 450 UCGCAUAAUCUGCAUGGU 1340- 901319. 1701195. AUGCGA 1362 1701196. GAUGU 1362 1 1 1 AD- A- 322 UGAGUUAAACGAACG 1692- A- 451 UAAGUACGUUCGUUUAAC 1690- 901341. 1701239. UACUUA 1712 1701240. UCAAG 1712 1 1 1 AD- A- 323 GAAAGUGUUUUAUA 2946- A- 452 UACCGUAUAUAAAACACU 2944- 901422. 1701401. UACGGUA 2966 1701402. UUCUC 2966 1 1 1 AD- A- 324 ACAGUCACUAGCUUA 3164- A- 453 UCAAGATAAGCUAGUGAC 3162- 901385. 1701327. UCUUGA 3184 1701328. UGUCA 3184 1 1 1 AD- A- 325 GUGCUACUGUUUAUC 3481- A- 454 UUUACGGAUAAACAGUA 3479- 901391. 1701339. CGUAAA 3501 1701340. GCACCA 3501 1 1 1 AD- A- 326 CCUGCAAAAACACAG 1652- A- 455 UCGAGUCUGUGUUUUUGC 1650- 901329. 1701215. ACUCGA 1672 1701216. AGGAA 1672 1 1 1 AD- A- 327 CAAAAACACAGACUC 1656- A- 456 UAACGCGAGUCUGUGUUU 1654- 901331. 1701219. GCGUUA 1676 1701220. UUGCA 1676 1 1 1 AD- A- 328 UGCUGUGGACUUGAG 2221- A- 457 UCCCAACUCAAGUCCACA 2219- 901368. 1701293. UUGGGA 2241 1701294. GCAGU 2241 1 1 1 AD- A- 329 GUGCUAAUGUUAUUG 2182- A- 458 UGACACCAAUAACAUUAG 2180- 901364. 1701285. GUGUCA 2202 1701286. CACUG 2202 1 1 1 AD- A- 330 UGGUGCUACUGUUUA 3479- A- 459 UACGGATAAACAGUAGCA 3477- 901389. 1701335. UCCGUA 3499 1701336. CCAAU 3499 1 1 1 AD- A- 331 AGAAAGUGUUUUAU 2945- A- 460 UCCGUATAUAAAACACUU 2943- 901421. 1701399. AUACGGA 2965 1701400. UCUCU 2965 1 1 1 AD- A- 332 AACUAUUUAUGAGAU 3062- A- 461 UGAUACAUCUCAUAAAUA 3060- 901380. 1701317. GUAUCA 3082 1701318. GUUGA 3082 1 1 1 AD- A- 333 AGUUAAACGAACGUA 1694- A- 462 UGCAAGTACGUUCGUUUA 1692- 901343. 1701243. CUUGCA 1714 1701244. ACUCA 1714 1 1 1 AD- A- 334 CAUCUUCAAGCCAUC 1251- A- 463 UCACAGGAUGGCUUGAAG 1249- 901317. 1701191. CUGUGA 1271 1701192. AUGUA 1271 1 1 1 AD- A- 335 UUUUUUUUCAGUAUU 3027- A- 464 UCCAAGAAUACUGAAAAA 3025- 901424. 1701405. CUUGGA 3047 1701406. AAACC 3047 1 1 1 AD- A- 336 UUAUCCGUAAUAAUU 3491- A- 465 UCCCACAAUUAUUACGGA 3489- 901431. 1701419. GUGGGA 3511 1701420. UAAAC 3511 1 1 1 AD- A- 337 GGUACUUAUUUAAUA 2963- A- 466 UAGGGATAUUAAAUAAG 2961- 901378. 1701313. UCCCUA 2983 1701314. UACCGU 2983 1 1 1 AD- A- 338 CACGUCUUUGUCUCU 3527- A- 467 UGCACUAGAGACAAAGAC 3525- 901434. 1701425. AGUGCA 3547 1701426. GUGAU 3547 1 1 1 AD- A- 339 UGCAAAAACACAGAC 1654- A- 468 UCGCGAGUCUGUGUUUUU 1652- 901412. 1701381. UCGCGA 1674 1701382. GCAGG 1674 1 1 1 AD- A- 340 ACUAUUUAUGAGAUG 3063- A- 469 UAGAUACAUCUCAUAAAU 3061- 901426. 1701409. UAUCUA 3083 1701410. AGUUG 3083 1 1 1 AD- A- 341 AGGGGCAAAAACGAA 1484- A- 470 UGCGCUTUCGUUUUUGCC 1482- 901322. 1701201. AGCGCA 1504 1701202. CCUUU 1504 1 1 1 AD- A- 342 UUGCUCUCUUAUUUG 3094- A- 471 UCGGUACAAAUAAGAGA 3092- 901381. 1701319. UACCGA 3114 1701320. GCAAGA 3114 1 1 1 AD- A- 343 AUUUGUUUGUACAAG 1616- A- 472 UCGGAUCUUGUACAAACA 1614- 901324. 1701205. AUCCGA 1636 1701206. AAUGC 1636 1 1 1 AD- A- 344 UUGCAGAUGUGACAA 1710- A- 473 UUCGGCTUGUCACAUCUG 1708- 901347. 1701251. GCCGAA 1730 1701252. CAAGU 1730 1 1 1 AD- A- 345 CAACUAUUUAUGAGA 3061- A- 474 UAUACATCUCAUAAAUAG 3059- 901379. 1701315. UGUAUA 3081 1701316. UUGAA 3081 1 1 1 AD- A- 346 AAUUCUACAUACUAA 3419- A- 475 UGAGAUTUAGUAUGUAG 3417- 901428. 1701413. AUCUCA 3439 1701414. AAUUCU 3439 1 1 1 AD- A- 347 AUGUCCUCACACCAU 2782- A- 476 UUUUCAAUGGUGUGAGG 2780- 901371. 1701299. UGAAAA 2802 1701300. ACAUAG 2802 1 1 1 AD- A- 348 UUGUUUGUACAAGAU 1618- A- 477 UUGCGGAUCUUGUACAAA 1616- 901408. 1701373. CCGCAA 1638 1701374. CAAAU 1638 1 1 1 AD- A- 349 UAAUCCAGAAACCUG 1870- A- 478 UCAUUUCAGGUUUCUGGA 1868- 901417. 1701391. AAAUGA 1890 1701392. UUAAG 1890 1 1 1 AD- A- 350 GAAAAAAAAUCAGUU 1456- A- 479 UCCUCGAACUGAUUUUUU 1454- 901400. 1701357. CGAGGA 1476 1701358. UUCUU 1476 1 1 1 AD- A- 351 GGGCAAAAACGAAAG 1486- A- 480 UUUGCGCUUUCGUUUUUG 1484- 901323. 1701203. CGCAAA 1506 1701204. CCCCU 1506 1 1 1 AD- A- 352 UGAAGUUCAUGGAUG 1160- A- 481 UAUAGACAUCCAUGAACU 1158- 901316. 1701189. UCUAUA 1180 1701190. UCACC 1180 1 1 1 AD- A- 353 CACGAAGUGGUGAAG 1150- A- 482 UAUGAACUUCACCACUUC 1148- 901315. 1701187. UUCAUA 1170 1701188. GUGAU 1170 1 1 1 AD- A- 354 ACGUCUUUGUCUCUA 3528- A- 483 UUGCACTAGAGACAAAGA 3526- 901395. 1701347. GUGCAA 3548 1701348. CGUGA 3548 1 1 1 AD- A- 355 AACAUCACCAUGCAG 1339- A- 484 UAUAAUCUGCAUGGUGA 1337- 901318. 1701193. AUUAUA 1359 1701194. UGUUGG 1359 1 1 1 AD- A- 356 GGUGCUACUGUUUAU 3480- A- 485 UUACGGAUAAACAGUAGC 3478- 901390. 1701337. CCGUAA 3500 1701338. ACCAA 3500 1 1 1 AD- A- 357 AAAUAGACAUUGCUA 3362- A- 486 UCAGAATAGCAAUGUCUA 3360- 901387. 1701331. UUCUGA 3382 1701332. UUUUA 3382 1 1 1 AD- A- 358 UUCCCCAAAUCACUG  278- A- 487 UAUCCACAGUGAUUUGGG  276- 901307. 1701171. UGGAUA  298 1701172. GAAGU  298 1 1 1 AD- A- 359 GAUCCGCAGACGUGU 1630- A- 488 UCAUUUACACGUCUGCGG 1628- 901410. 1701377. AAAUGA 1650 1701378. AUCUU 1650 1 1 1 AD- A- 360 UUAACAUCACGUCUU 3520- A- 489 UAGACAAAGACGUGAUG 3518- 901433. 1701423. UGUCUA 3540 1701424. UUAAUA 3540 1 1 1 AD- A- 361 AAAGUGAGUGACCUG  415- A- 490 UAAAAGCAGGUCACUCAC  413- 901308. 1701173. CUUUUA  435 1701174. UUUGC  435 1 1 1 AD- A- 362 AAAAACACAGACUCG 1657- A- 491 UCAACGCGAGUCUGUGUU 1655- 901414. 1701385. CGUUGA 1677 1701386. UUUGC 1677 1 1 1 AD- A- 363 CGUCGCACUGAAACU  645- A- 492 UCGAAAAGUUUCAGUGCG  643- 901309. 1701175. UUUCGA  665 1701176. ACGCC  665 1 1 1 AD- A- 364 AACAGUGCUAAUGUU 2178- A- 493 UCCAAUAACAUUAGCACU 2176- 901362. 1701281. AUUGGA 2198 1701282. GUUAA 2198 1 1 1 AD- A- 365 UUCGUCCAACUUCUG  661- A- 494 UCAGCCCAGAAGUUGGAC  659- 901397. 1701351. GGCUGA  681 1701352. GAAAA  681 1 1 1 AD- A- 366 AUUGGUGUCUUCACU 2193- A- 495 UCAUCCAGUGAAGACACC 2191- 901419. 1701395. GGAUGA 2213 1701396. AAUAA 2213 1 1 1 AD- A- 367 GCAAAAACACAGACU 1655- A- 496 UACGCGAGUCUGUGUUUU 1653- 901413. 1701383. CGCGUA 1675 1701384. UGCAG 1675 1 1 1 AD- A- 368 AAAAAUCAGUUCGAG 1460- A- 497 UCUUUCCUCGAACUGAUU 1458- 901401. 1701359. GAAAGA 1480 1701360. UUUUU 1480 1 1 1 AD- A- 369 CAGACGUGUAAAUGU 1636- A- 498 UCAGGAACAUUUACACGU 1634- 901411. 1701379. UCCUGA 1656 1701380. CUGCG 1656 1 1 1 AD- A- 370 UGUCCUCACACCAUU 2783- A- 499 UGUUUCAAUGGUGUGAG 2781- 901372. 1701301. GAAACA 2803 1701302. GACAUA 2803 1 1 1 AD- A- 371 UUUUUUUCAGUAUUC 3028- A- 500 UACCAAGAAUACUGAAAA 3026- 901425. 1701407. UUGGUA 3048 1701408. AAAAC 3048 1 1 1 AD- A- 372 AGAUCCGCAGACGUG 1629- A- 501 UAUUUACACGUCUGCGGA 1627- 901409. 1701375. UAAAUA 1649 1701376. UCUUG 1649 1 1 1 AD- A- 373 CCCUCUUGGAAUUGG 1978- A- 502 UCGAAUCCAAUUCCAAGA 1976- 901418. 1701393. AUUCGA 1998 1701394. GGGAC 1998 1 1 1 AD- A- 374 GAUAUUAACAUCACG 3516- A- 503 UAAAGACGUGAUGUUAA 3514- 901393. 1701343. UCUUUA 3536 1701344. UAUCUU 3536 1 1 1 AD- A- 375 UUGGUGCUACUGUUU 3478- A- 504 UCGGAUAAACAGUAGCAC 3476- 901388. 1701333. AUCCGA 3498 1701334. CAAUA 3498 1 1 1 AD- A- 376 AAGGGGCAAAAACGA 1483- A- 505 UCGCUUTCGUUUUUGCCC 1481- 901404. 1701365. AAGCGA 1503 1701366. cuuuc 1503 1 1 1 AD- A- 377 CUUGCAGAUGUGACA 1709- A- 506 UCGGCUTGUCACAUCUGC 1707- 901346. 1701249. AGCCGA 1729 1701250. AAGUA 1729 1 1 1 AD- A- 378 AAAUCAGUUCGAGGA 1462- A- 507 UCCCUUTCCUCGAACUGA 1460- 901403. 1701363. AAGGGA 1482 1701364. UUUUU 1482 1 1 1 AD- A- 379 ACUUUUCGUCCAACU  657- A- 508 UCCAGAAGUUGGACGAAA  655- 901396. 1701349. UCUGGA  677 1701350. AGUUU  677 1 1 1 AD- A- 380 AUUAACAUCACGUCU 3519- A- 509 UGACAAAGACGUGAUGU 3517- 901432. 1701421. UUGUCA 3539 1701422. UAAUAU 3539 1 1 1 AD- A- 381 CGUCUUUGUCUCUAG 3529- A- 510 UCUGCACUAGAGACAAAG 3527- 901435. 1701427. UGCAGA 3549 1701428. ACGUG 3549 1 1 1 AD- A- 382 AACACAGACUCGCGU 1660- A- 511 UUUGCAACGCGAGUCUGU 1658- 901416. 1701389. UGCAAA 1680 1701390. GUUUU 1680 1 1 1 AD- A- 383 AUAUUAACAUCACGU 3517- A- 512 UCAAAGACGUGAUGUUA 3515- 901394. 1701345. CUUUGA 3537 1701346. AUAUCU 3537 1 1 1 AD- A- 384 GUUUAUCCGUAAUAA 3489- A- 513 UCACAATUAUUACGGAUA 3487- 901429. 1701415. UUGUGA 3509 1701416. AACAG 3509 1 1 1 AD- A- 385 AAAAUCAGUUCGAGG 1461- A- 514 UCCUUUCCUCGAACUGAU 1459- 901402. 1701361. AAAGGA 1481 1701362. UUUUU 1481 1 1 1 AD- A- 386 UUUAUCCGUAAUAAU 3490- A- 515 UCCACAAUUAUUACGGAU 3488- 901430. 1701417. UGUGGA 3510 1701418. AAACA 3510 1 1 1 AD- A- 387 AGGACAUUGCUGUGC 2518- A- 516 UCCAAAGCACAGCAAUGU 2516- 901369. 1701295. UUUGGA 2538 1701296. CCUGA 2538 1 1 1

TABLE 3A Exemplary Human VEGF-A siRNA Modified Single Strands and Duplex Sequences Anti- SEQ SEQ ID Sense SEQ sense ID NO: NO: Duplex Oligo ID NO: Oligo (Anti- mRNA target (mRNA Name Name (Sense) Sense Sequence Name sense) Antisense Sequence sequence target) AD- A- 517 ascsuga(Uhd)AfcAfGfAfac A- 647 VPusAfsucgAfuCfGfu AGACUGAUACAG 4353 953340.1 1701261. gaucgauaL96 1068804. ucuGfuAfucaguscsu AACGAUCGAUA 1 1 AD- A- 518 asasaga(Chd)UfgAfUfAfca A- 648 VPusAfsucgUfuCfUfg GGAAAGACUGAU 4354 953336.1 1701253. gaacgauaL96 1068796. uauCfaGfucuuuscsc ACAGAACGAUC 1 1 AD- A- 519 gsasgaa(Ahd)GfuGfUfUfuu A- 649 VPusCfsguaUfaUfAfaa AAGAGAAAGUGU 4355 953363.1 1701307. auauacgaL96 1070290. acAfcUfuucucsusu UUUAUAUACGG 1 1 AD- A- 520 asgsacu(Ghd)AfuAfCfAfga A- 650 VPusCfsgauCfgUfUfcu AAAGACUGAUAC 4356 953338.1 1701257. acgaucgaL96 1068800. guAfuCfagucususu AGAACGAUCGA 1 1 AD- A- 521 csasacu(Ahd)UfuUfAfUfga A- 651 VPusAfsuacAfuCfUfca UUCAACUAUUUA 4357 953367.1 1701315. gauguauaL96 1070376. uaAfaUfaguugsasa UGAGAUGUAUC 1 1 AD- A- 522 asasgac(Uhd)GfaUfAfCfag A- 652 VPusGfsaucGfuUfCfug GAAAGACUGAUA 4358 953337.1 1701255. aacgaucaL96 1068798. uaUfcAfgucuususc CAGAACGAUCG 1 1 AD- A- 523 asusaca(Ghd)AfaCfGfAfuc A- 653 VPusCfsuguAfuCfGfau UGAUACAGAACG 4359 953342.1 1701265. gauacagaL96 1068812. cgUfuCfuguauscsa AUCGAUACAGA 1 1 AD- A- 524 asascag(Uhd)GfcUfAfAfug A- 654 VPusCfscaaUfaAfCfau UUAACAGUGCUA 4360 953350.1 1701281. uuauuggaL96 1069342. uaGfcAfcuguusasa AUGUUAUUGGU 1 1 AD- A- 525 gsusgcu(Ahd)AfuGfUfUfau A- 655 VPusGfsacaCfcAfAfua CAGUGCUAAUGU 4361 953352.1 1701285. uggugucaL96 1069350. acAfuUfagcacsusg UAUUGGUGUCU 1 1 AD- A- 526 asascua(Uhd)UfuAfUfGfag A- 656 VPusGfsauaCfaUfCfuc UCAACUAUUUAU 4362 953368.1 1701317. auguaucaL96 1070378. auAfaAfuaguusgsa GAGAUGUAUCU 1 1 AD- A- 527 csasgaa(Chd)AfgUfCfCfuu A- 657 VPusCfsuggAfuUfAfa GACAGAACAGUCC 4363 953344.1 1701269. aauccagaL96 1068918. ggaCfuGfuucugsusc UUAAUCCAGA 1 1 AD- A- 528 gsascug(Ahd)UfaCfAfGfaa A- 658 VPusUfscgaUfcGfUfuc AAGACUGAUACA 4364 953339.1 1701259. cgaucgaaL96 1068802. ugUfaUfcagucsusu GAACGAUCGAU 1 1 AD- A- 529 ascsagc(Ahd)CfaAfCfAfaa A- 659 VPusAfsuucAfcAfUfu CUACAGCACAACA 4365 953387.1 1701355. ugugaauaL96 1068170. uguUfgUfgcugusasg AAUGUGAAUG 1 1 AD- A- 530 asasaua(Ghd)AfcAfUfUfgc A- 660 VPusCfsagaAfuAfGfca UAAAAUAGACAU 4366 953375.1 1701331. uauucugaL96 1070792. auGfuCfuauuususa UGCUAUUCUGU 1 1 AD- A- 531 usasuug(Ghd)UfgUfCfUfuc A- 661 VPusAfsuccAfgUfGfaa GUUAUUGGUGUC 4367 953355.1 1701291. acuggauaL96 1069370. gaCfaCfcaauasasc UUCACUGGAUG 1 1 AD- A- 532 csusgau(Ahd)CfaGfAfAfcg A- 662 VPusUfsaucGfaUfCfgu GACUGAUACAGA 4368 953341.1 1701263. aucgauaaL96 1068806. ucUfgUfaucagsusc ACGAUCGAUAC 1 1 AD- A- 533 gscsucu(Chd)UfuAfUfUfug A- 663 VPusAfsccgGfuAfCfaa UUGCUCUCUUAUU 4369 953370.1 1701321. uaccgguaL96 1070446. auAfaGfagagcsasa UGUACCGGUU 1 1 AD- A- 534 csascca(Uhd)UfgAfAfAfcc A- 664 VPusAfsacuAfgUfGfg CACACCAUUGAAA 4370 953362.1 1701305. acuaguuaL96 1070096. uuuCfaAfuggugsusg CCACUAGUUC 1 1 AD- A- 535 gsgscag(Chd)UfuGfAfGfuu A- 665 VPusUfsucgUfuUfAfac GAGGCAGCUUGA 4371 953322.1 1701225. aaacgaaaL96 1068596. ucAfaGfcugccsusc GUUAAACGAAC 1 1 AD- A- 536 ususaaa(Chd)GfaAfCfGfua A- 666 VPusCfsugcAfaGfUfac AGUUAAACGAAC 4372 953332.1 1701245. cuugcagaL96 1068618. guUfcGfuuuaascsu GUACUUGCAGA 1 1 AD- A- 537 uscsggu(Ghd)AfcAfGfUfca A- 667 VPusAfsagcUfaGfUfga GAUCGGUGACAG 4373 953371.1 1701323. cuagcuuaL96 1070550. cuGfuCfaccgasusc UCACUAGCUUA 1 1 AD- A- 538 asgsuua(Ahd)AfcGfAfAfcg A- 668 VPusGfscaaGfuAfCfgu UGAGUUAAACGA 4374 953331.1 1701243. uacuugcaL96 1068614. ucGfuUfuaacuscsa ACGUACUUGCA 1 1 AD- A- 539 gscsagc(Uhd)UfgAfGfUfua A- 669 VPusGfsuucGfuUfUfaa AGGCAGCUUGAG 4375 953323.1 1701227. aacgaacaL96 1068598. cuCfaAfgcugcscsu UUAAACGAACG 1 1 AD- A- 540 asgsugc(Uhd)AfaUfGfUfua A- 670 VPusAfscacCfaAfUfaa ACAGUGCUAAUG 4376 953351.1 1701283. uugguguaL96 1069348. caUfuAfgcacusgsu UUAUUGGUGUC 1 1 AD- A- 541 csgsaag(Uhd)GfgUfGfAfag A- 671 VPusCfscauGfaAfCfuu CACGAAGUGGUG 4377 953386.1 1701353. uucauggaL96 1067728. caCfcAfcuucgsusg AAGUUCAUGGA 1 1 AD- A- 542 asasagc(Ahd)UfuUfGfUfuu A- 672 VPusCfsuugUfaCfAfaa AGAAAGCAUUUG 4378 953394.1 1701369. guacaagaL96 1068448. caAfaUfgcuuuscsu UUUGUACAAGA 1 1 AD- A- 543 asusguc(Chd)UfcAfCfAfcc A- 673 VPusUfsuucAfaUfGfg CUAUGUCCUCACA 4379 953359.1 1701299. auugaaaaL96 1070078. uguGfaGfgacausasg CCAUUGAAAC 1 1 AD- A- 544 usgsagu(Uhd)AfaAfCfGfaa A- 674 VPusAfsaguAfcGfUfuc CUUGAGUUAAAC 4380 953329.1 1701239. cguacuuaL96 1068610. guUfuAfacucasasg GAACGUACUUG 1 1 AD- A- 545 ascsacc(Ahd)UfuGfAfAfac A- 675 VPusAfscuaGfuGfGfu UCACACCAUUGAA 4381 953361.1 1701303. cacuaguaL96 1070094. uucAfaUfggugusgsa ACCACUAGUU 1 1 AD- A- 546 csasaaa(Ahd)CfaCfAfGfac A- 676 VPusAfsacgCfgAfGfuc UGCAAAAACACAG 4382 953319.1 1701219. ucgcguuaL96 1068538. ugUfgUfuuuugscsa ACUCGCGUUG 1 1 AD- A- 547 usgsucc(Uhd)CfaCfAfCfca A- 677 VPusGfsuuuCfaAfUfg UAUGUCCUCACAC 4383 953360.1 1701301. uugaaacaL96 1070080. gugUfgAfggacasusa CAUUGAAACC 1 1 AD- A- 548 csasgcu(Uhd)GfaGfUfUfaa A- 678 VPusCfsguuCfgUfUfua GGCAGCUUGAGU 4384 953324.1 1701229. acgaacgaL96 1068600. acUfcAfagcugscsc UAAACGAACGU 1 1 AD- A- 549 gsgsugc(Uhd)AfcUfGfUfuu A- 679 VPusUfsacgGfaUfAfaa UUGGUGCUACUG 4385 953378.1 1701337. auccguaaL96 1070874. caGfuAfgcaccsasa UUUAUCCGUAA 1 1 AD- A- 550 ususgcu(Chd)UfcUfUfAfuu A- 680 VPusCfsgguAfcAfAfau UCUUGCUCUCUUA 4386 953369.1 1701319. uguaccgaL96 1070442. aaGfaGfagcaasgsa UUUGUACCGG 1 1 AD- A- 551 uscsuug(Chd)UfgCfUfAfaa A- 681 VPusUfscggUfgAfUfu UUUCUUGCUGCUA 4387 953347.1 1701275. ucaccgaaL96 1069188. uagCfaGfcaagasasa AAUCACCGAG 1 1 AD- A- 552 csgsgua(Chd)UfuAfUfUfua A- 682 VPusGfsggaUfaUfUfaa UACGGUACUUAU 4388 953365.1 1701311. auaucccaL96 1070326. auAfaGfuaccgsusa UUAAUAUCCCU 1 1 AD- A- 553 asasaau(Ahd)GfaCfAfUfug A- 683 VPusAfsgaaUfaGfCfaa AUAAAAUAGACA 4389 953374.1 1701329. cuauucuaL96 1070790. ugUfcUfauuuusasu UUGCUAUUCUG 1 1 AD- A- 554 ascsuuu(Uhd)CfgUfCfCfaa A- 684 VPusCfscagAfaGfUfug AAACUUUUCGUCC 4390 953384.1 1701349. cuucuggaL96 1067266. gaCfgAfaaagususu AACUUCUGGG 1 1 AD- A- 555 ususggu(Ghd)CfuAfCfUfgu A- 685 VPusCfsggaUfaAfAfca UAUUGGUGCUAC 4391 953376.1 1701333. uuauccgaL96 1070870. guAfgCfaccaasusa UGUUUAUCCGU 1 1 AD- A- 556 gsusuau(Uhd)GfgUfGfUfcu A- 686 VPusCfscagUfgAfAfga AUGUUAUUGGUG 4392 953354.1 1701289. ucacuggaL96 1069366. caCfcAfauaacsasu UCUUCACUGGA 1 1 AD- A- 557 ususcgu(Chd)CfaAfCfUfuc A- 687 VPusCfsagcCfcAfGfaa UUUUCGUCCAACU 4393 953385.1 1701351. ugggcugaL96 1067274. guUfgGfacgaasasa UCUGGGCUGU 1 1 AD- A- 558 ususcuu(Ghd)CfuGfCfUfaa A- 688 VPusCfsgguGfaUfUfua UUUUCUUGCUGCU 4394 953346.1 1701273. aucaccgaL96 1069186. gcAfgCfaagaasasa AAAUCACCGA 1 1 AD- A- 559 gsgsuac(Uhd)UfaUfUfUfaa A- 689 VPusAfsgggAfuAfUfu ACGGUACUUAUU 4395 953366.1 1701313. uaucccuaL96 1070328. aaaUfaAfguaccsgsu UAAUAUCCCUU 1 1 AD- A- 560 asusauu(Ahd)AfcAfUfCfac A- 690 VPusCfsaaaGfaCfGfug AGAUAUUAACAU 4396 953382.1 1701345. gucuuugaL96 1070912. auGfuUfaauauscsu CACGUCUUUGU 1 1 AD- A- 561 gsasggc(Ahd)GfcUfUfGfag A- 691 VPusCfsguuUfaAfCfuc GCGAGGCAGCUUG 4397 953320.1 1701221. uuaaacgaL96 1068592. aaGfcUfgccucsgsc AGUUAAACGA 1 1 AD- A- 562 gsusgcu(Ahd)CfuGfUfUfua A- 692 VPusUfsuacGfgAfUfaa UGGUGCUACUGU 4398 953379.1 1701339. uccguaaaL96 1070876. acAfgUfagcacscsa UUAUCCGUAAU 1 1 AD- A- 563 asgsgca(Ghd)CfuUfGfAfgu A- 693 VPusUfscguUfuAfAfc CGAGGCAGCUUGA 4399 953321.1 1701223. uaaacgaaL96 1068594. ucaAfgCfugccuscsg GUUAAACGAA 1 1 AD- A- 564 usgsgug(Chd)UfaCfUfGfuu A- 694 VPusAfscggAfuAfAfac AUUGGUGCUACU 4400 953377.1 1701335. uauccguaL96 1070872. agUfaGfcaccasasu GUUUAUCCGUA 1 1 AD- A- 565 asasggg(Ghd)CfaAfAfAfac A- 695 VPusCfsgcuUfuCfGfuu GAAAGGGGCAAA 4401 953392.1 1701365. gaaagcgaL96 1700876. uuUfgCfcccuususc AACGAAAGCGC 1 1 AD- A- 566 ascsagu(Chd)AfcUfAfGfcu A- 696 VPusCfsaagAfuAfAfgc UGACAGUCACUAG 4402 953373.1 1701327. uaucuugaL96 1070562. uaGfuGfacuguscsa CUUAUCUUGA 1 1 AD- A- 567 ascsggu(Ahd)CfuUfAfUfuu A- 697 VPusGfsgauAfuUfAfaa AUACGGUACUUA 4403 953364.1 1701309. aauauccaL96 1070324. uaAfgUfaccgusasu UUUAAUAUCCC 1 1 AD- A- 568 gsasguu(Ahd)AfaCfGfAfac A- 698 VPusCfsaagUfaCfGfuu UUGAGUUAAACG 4404 953330.1 1701241. guacuugaL96 1068612. cgUfuUfaacucsasa AACGUACUUGC 1 1 AD- A- 569 usgsuua(Uhd)UfgGfUfGfuc A- 699 VPusCfsaguGfaAfGfac AAUGUUAUUGGU 4405 953353.1 1701287. uucacugaL96 1069364. acCfaAfuaacasusu GUCUUCACUGG 1 1 AD- A- 570 csgsaca(Ghd)AfaCfAfGfuc A- 700 VPusGfsauuAfaGfGfac AUCGACAGAACAG 4406 953343.1 1701267. cuuaaucaL96 1068912. ugUfuCfugucgsasu UCCUUAAUCC 1 1 AD- A- 571 asasaau(Chd)AfgUfUfCfga A- 701 VPusCfscuuUfcCfUfcg AAAAAAUCAGUU 4407 953390.1 1701361. ggaaaggaL96 1700873. aaCfuGfauuuususu CGAGGAAAGGG 1 1 AD- A- 572 csusugg(Ahd)AfuUfGfGfau A- 702 VPusAfsuggCfgAfAfu CUCUUGGAAUUG 4408 953345.1 1701271. ucgccauaL96 1069132. ccaAfuUfccaagsasg GAUUCGCCAUU 1 1 AD- A- 573 asgsaga(Ahd)GfaGfAfCfac A- 703 VPusCfsaacAfaUfGfug GAAGAGAAGAGA 4409 953358.1 1701297. auuguugaL96 1069954. ucUfcUfucucususc CACAUUGUUGG 1 1 AD- A- 574 ascsguc(Uhd)UfuGfUfCfuc A- 704 VPusUfsgcaCfuAfGfag UCACGUCUUUGUC 4410 953383.1 1701347. uagugcaaL96 1070934. acAfaAfgacgusgsa UCUAGUGCAG 1 1 AD- A- 575 usgsaca(Ghd)UfcAfCfUfag A- 705 VPusAfsgauAfaGfCfua GGUGACAGUCACU 4411 953372.1 1701325. cuuaucuaL96 1070558. guGfaCfugucascsc AGCUUAUCUU 1 1 AD- A- 576 ususgag(Uhd)UfaAfAfCfga A- 706 VPusAfsguaCfgUfUfcg GCUUGAGUUAAA 4412 953328.1 1701237. acguacuaL96 1068608. uuUfaAfcucaasgsc CGAACGUACUU 1 1 AD- A- 577 gsasaag(Chd)AfuUfUfGfuu A- 707 VPusUfsuguAfcAfAfac GAGAAAGCAUUU 4413 953393.1 1701367. uguacaaaL96 1068446. aaAfuGfcuuucsusc GUUUGUACAAG 1 1 AD- A- 578 asuscac(Chd)AfuGfCfAfga A- 708 VPusCfsgcaUfaAfUfcu ACAUCACCAUGCA 4414 953307.1 1701195. uuaugcgaL96 1068040. gcAfuGfgugausgsu GAUUAUGCGG 1 1 AD- A- 579 csascca(Uhd)GfcAfGfAfuu A- 709 VPusUfsccgCfaUfAfau AUCACCAUGCAGA 4415 953308.1 1701197. augcggaaL96 1068044. cuGfcAfuggugsasu UUAUGCGGAU 1 1 AD- A- 580 csusuga(Ghd)UfuAfAfAfcg A- 710 VPusGfsuacGfuUfCfgu AGCUUGAGUUAA 4416 953327.1 1701235. aacguacaL96 1068606. uuAfaCfucaagscsu ACGAACGUACU 1 1 AD- A- 581 ususgca(Ghd)AfuGfUfGfac A- 711 VPusUfscggCfuUfGfuc ACUUGCAGAUGU 4417 953335.1 1701251. aagccgaaL96 1068646. acAfuCfugcaasgsu GACAAGCCGAG 1 1 AD- A- 582 ascsuau(Uhd)UfaUfGfAfga A- 712 VPusAfsgauAfcAfUfcu CAACUAUUUAUG 4418 953414.1 1701409. uguaucuaL96 1070380. caUfaAfauagususg AGAUGUAUCUU 1 1 AD- A- 583 ususuuu(Uhd)UfuCfAfGfua A- 713 VPusCfscaaGfaAfUfac GGUUUUUUUUCA 4419 953412.1 1701405. uucuuggaL96 1700897. ugAfaAfaaaaascsc GUAUUCUUGGU 1 1 AD- A- 584 gsusuuu(Ahd)UfaUfAfCfgg A- 714 VPusAfsuaaGfuAfCfcg GUGUUUUAUAUA 4420 953411.1 1701403. uacuuauaL96 1070306. uaUfaUfaaaacsasc CGGUACUUAUU 1 1 AD- A- 585 gsasaag(Uhd)GfuUfUfUfau A- 715 VPusAfsccgUfaUfAfua GAGAAAGUGUUU 4421 953410.1 1701401. auacgguaL96 1070294. aaAfcAfcuuucsusc UAUAUACGGUA 1 1 AD- A- 586 uscsacu(Ghd)GfaUfGfUfau A- 716 VPusCfsaguCfaAfAfua CUUCACUGGAUGU 4422 953408.1 1701397. uugacugaL96 1069392. caUfcCfagugasasg AUUUGACUGC 1 1 AD- A- 587 gscsuug(Ahd)GfuUfAfAfac A- 717 VPusUfsacgUfuCfGfuu CAGCUUGAGUUA 4423 953326.1 1701233. gaacguaaL96 1068604. uaAfcUfcaagcsusg AACGAACGUAC 1 1 AD- A- 588 cscsucc(Ghd)AfaAfCfCfau A- 718 VPusAfsaagUfuCfAfug GGCCUCCGAAACC 4424 953300.1 1701181. gaacuuuaL96 1067482. guUfuCfggaggscsc AUGAACUUUC 1 1 AD- A- 589 asasaaa(Uhd)CfaGfUfUfcg A- 719 VPusCfsuuuCfcUfCfga AAAAAAAUCAGU 4425 953389.1 1701359. aggaaagaL96 1700871. acUfgAfuuuuususu UCGAGGAAAGG 1 1 AD- A- 590 gsasgaa(Uhd)UfcUfAfCfau A- 720 VPusAfsuuuAfgUfAfu UAGAGAAUUCUA 4426 953415.1 1701411. acuaaauaL96 1070816. guaGfaAfuucucsusa CAUACUAAAUC 1 1 AD- A- 591 asgsauu(Ahd)UfgCfGfGfau A- 721 VPusAfsgguUfuGfAfu GCAGAUUAUGCG 4427 953309.1 1701199. caaaccuaL96 1068060. ccgCfaUfaaucusgsc GAUCAAACCUC 1 1 AD- A- 592 asasauc(Ahd)GfuUfCfGfag A- 722 VPusCfsccuUfuCfCfuc AAAAAUCAGUUC 4428 953391.1 1701363. gaaagggaL96 1068240. gaAfcUfgauuususu GAGGAAAGGGA 1 1 AD- A- 593 gscsauu(Uhd)GfuUfUfGfua A- 723 VPusGfsaucUfuGfUfac AAGCAUUUGUUU 4429 953395.1 1701371. caagaucaL96 1068454. aaAfcAfaaugcsusu GUACAAGAUCC 1 1 AD- A- 594 csascga(Ahd)GfuGfGfUfga A- 724 VPusAfsugaAfcUfUfca AUCACGAAGUGG 4430 953303.1 1701187. aguucauaL96 1067724. ccAfcUfucgugsasu UGAAGUUCAUG 1 1 AD- A- 595 usasauc(Chd)AfgAfAfAfcc A- 725 VPusCfsauuUfcAfGfgu CUUAAUCCAGAAA 4431 953405.1 1701391. ugaaaugaL96 1068942. uuCfuGfgauuasasg CCUGAAAUGA 1 1 AD- A- 596 csasucu(Uhd)CfaAfGfCfca A- 726 VPusCfsacaGfgAfUfgg UACAUCUUCAAGC 4432 953305.1 1701191. uccugugaL96 1067926. cuUfgAfagaugsusa CAUCCUGUGU 1 1 AD- A- 597 usgscua(Chd)UfgUfUfUfau A- 727 VPusAfsuuaCfgGfAfua GGUGCUACUGUU 4433 953380.1 1701341. ccguaauaL96 1070878. aaCfaGfuagcascsc UAUCCGUAAUA 1 1 AD- A- 598 ususgcu(Ghd)CfuAfAfAfuc A- 728 VPusGfscucGfgUfGfau UCUUGCUGCUAAA 4434 953349.1 1701279. accgagcaL96 1069192. uuAfgCfagcaasgsa UCACCGAGCC 1 1 AD- A- 599 gsasuau(Uhd)AfaCfAfUfca A- 729 VPusAfsaagAfcGfUfga AAGAUAUUAACA 4435 953381.1 1701343. cgucuuuaL96 1070910. ugUfuAfauaucsusu UCACGUCUUUG 1 1 AD- A- 600 csusgca(Ahd)AfaAfCfAfca A- 730 VPusGfscgaGfuCfUfgu UCCUGCAAAAACA 4436 953318.1 1701217. gacucgcaL96 1068532. guUfuUfugcagsgsa CAGACUCGCG 1 1 AD- A- 601 csusugc(Uhd)GfcUfAfAfau A- 731 VPusCfsucgGfuGfAfu UUCUUGCUGCUAA 4437 953348.1 1701277. caccgagaL96 1069190. uuaGfcAfgcaagsasa AUCACCGAGC 1 1 AD- A- 602 asgsaaa(Ghd)UfgUfUfUfua A- 732 VPusCfscguAfuAfUfaa AGAGAAAGUGUU 4438 953409.1 1701399. uauacggaL96 1070292. aaCfaCfuuucuscsu UUAUAUACGGU 1 1 AD- A- 603 asascau(Chd)AfcCfAfUfgc A- 733 VPusAfsuaaUfcUfGfca CCAACAUCACCAU 4439 953306.1 1701193. agauuauaL96 1068034. ugGfuGfauguusgsg GCAGAUUAUG 1 1 AD- A- 604 csgscag(Ahd)CfgUfGfUfaa A- 734 VPusGfsgaaCfaUfUfua UCCGCAGACGUGU 4440 953316.1 1701213. auguuccaL96 1068494. caCfgUfcugcgsgsa AAAUGUUCCU 1 1 AD- A- 605 asgscuu(Ghd)AfgUfUfAfaa A- 735 VPusAfscguUfcGfUfu GCAGCUUGAGUU 4441 953325.1 1701231. cgaacguaL96 1068602. uaaCfuCfaagcusgsc AAACGAACGUA 1 1 AD- A- 606 gscsacu(Ghd)AfaAfCfUfuu A- 736 VPusUfsggaCfgAfAfaa UCGCACUGAAACU 4442 953299.1 1701179. ucguccaaL96 1067250. guUfuCfagugcsgsa UUUCGUCCAA 1 1 AD- A- 607 asasuuc(Uhd)AfcAfUfAfcu A- 737 VPusGfsagaUfuUfAfg AGAAUUCUACAU 4443 953416.1 1701413. aaaucucaL96 1070822. uauGfuAfgaauuscsu ACUAAAUCUCU 1 1 AD- A- 608 cscsgca(Ghd)AfcGfUfGfua A- 738 VPusGfsaacAfuUfUfac AUCCGCAGACGUG 4444 953315.1 1701211. aauguucaL96 1068492. acGfuCfugcggsasu UAAAUGUUCC 1 1 AD- A- 609 uscscgc(Ahd)GfaCfGfUfgu A- 739 VPusAfsacaUfuUfAfca GAUCCGCAGACGU 4445 953314.1 1701209. aaauguuaL96 1068490. cgUfcUfgcggasusc GUAAAUGUUC 1 1 AD- A- 610 csgscac(Uhd)GfaAfAfCfuu A- 740 VPusGfsgacGfaAfAfag GUCGCACUGAAAC 4446 953298.1 1701177. uucguccaL96 1067248. uuUfcAfgugcgsasc UUUUCGUCCA 1 1 AD- A- 611 cscscuc(Uhd)UfgGfAfAfuu A- 741 VPusCfsgaaUfcCfAfau GUCCCUCUUGGAA 4447 953406.1 1701393. ggauucgaL96 1069124. ucCfaAfgagggsasc UUGGAUUCGC 1 1 AD- A- 612 csasgac(Ghd)UfgUfAfAfau A- 742 VPusCfsaggAfaCfAfuu CGCAGACGUGUAA 4448 953399.1 1701379. guuccugaL96 1068498. uaCfaCfgucugscsg AUGUUCCUGC 1 1 AD- A- 613 ascsgaa(Chd)GfuAfCfUfug A- 743 VPusAfscauCfuGfCfaa AAACGAACGUACU 4449 953333.1 1701247. cagauguaL96 1068626. guAfcGfuucgususu UGCAGAUGUG 1 1 AD- A- 614 asusccg(Chd)AfgAfCfGfug A- 744 VPusAfscauUfuAfCfac AGAUCCGCAGACG 4450 953313.1 1701207. uaaauguaL96 1068488. guCfuGfcggauscsu UGUAAAUGUU 1 1 AD- A- 615 csasgaa(Uhd)CfaUfCfAfcg A- 745 VPusAfsccaCfuUfCfgu GGCAGAAUCAUCA 4451 953302.1 1701185. aagugguaL96 1067706. gaUfgAfuucugscsc CGAAGUGGUG 1 1 AD- A- 616 cscsugc(Ahd)AfaAfAfCfac A- 746 VPusCfsgagUfcUfGfug UUCCUGCAAAAAC 4452 953317.1 1701215. agacucgaL96 1068530. uuUfuUfgcaggsasa ACAGACUCGC 1 1 AD- A- 617 asgsgac(Ahd)UfuGfCfUfgu A- 747 VPusCfscaaAfgCfAfca UCAGGACAUUGCU 4453 953357.1 1701295. gcuuuggaL96 1069740. gcAfaUfguccusgsa GUGCUUUGGG 1 1 AD- A- 618 gsgsgca(Ghd)AfaUfCfAfuc A- 748 VPusAfscuuCfgUfGfau GAGGGCAGAAUC 4454 953301.1 1701183. acgaaguaL96 1067700. gaUfuCfugcccsusc AUCACGAAGUG 1 1 AD- A- 619 usgsaag(Uhd)UfcAfUfGfga A- 749 VPusAfsuagAfcAfUfcc GGUGAAGUUCAU 4455 953304.1 1701189. ugucuauaL96 1067744. auGfaAfcuucascsc GGAUGUCUAUC 1 1 AD- A- 620 csgsucg(Chd)AfcUfGfAfaa A- 750 VPusCfsgaaAfaGfUfuu GGCGUCGCACUGA 4456 953297.1 1701175. cuuuucgaL96 1067242. caGfuGfcgacgscsc AACUUUUCGU 1 1 AD- A- 621 gsasaaa(Ahd)AfaAfUfCfag A- 751 VPusCfscucGfaAfCfug AAGAAAAAAAAU 4457 953388.1 1701357. uucgaggaL96 1700869. auUfuUfuuuucsusu CAGUUCGAGGA 1 1 AD- A- 622 asusugg(Uhd)GfuCfUfUfca A- 752 VPusCfsaucCfaGfUfga UUAUUGGUGUCU 4458 953407.1 1701395. cuggaugaL96 1069372. agAfcAfccaausasa UCACUGGAUGU 1 1 AD- A- 623 asgsauc(Chd)GfcAfGfAfcg A- 753 VPusAfsuuuAfcAfCfg CAAGAUCCGCAGA 4459 953397.1 1701375. uguaaauaL96 1068484. ucuGfcGfgaucususg CGUGUAAAUG 1 1 AD- A- 624 gsasucc(Ghd)CfaGfAfCfgu A- 754 VPusCfsauuUfaCfAfcg AAGAUCCGCAGAC 4460 953398.1 1701377. guaaaugaL96 1068486. ucUfgCfggaucsusu GUGUAAAUGU 1 1 AD- A- 625 ususguu(Uhd)GfuAfCfAfag A- 755 VPusUfsgcgGfaUfCfuu AUUUGUUUGUAC 4461 953396.1 1701373. auccgcaaL96 1068462. guAfcAfaacaasasu AAGAUCCGCAG 1 1 AD- A- 626 usgscug(Uhd)GfgAfCfUfug A- 756 VPusCfsccaAfcUfCfaa ACUGCUGUGGACU 4462 953356.1 1701293. aguugggaL96 1069428. guCfcAfcagcasgsu UGAGUUGGGA 1 1 AD- A- 627 csascgu(Chd)UfuUfGfUfcu A- 757 VPusGfscacUfaGfAfga AUCACGUCUUUGU 4463 953422.1 1701425. cuagugcaL96 1070932. caAfaGfacgugsasu CUCUAGUGCA 1 1 AD- A- 628 ususuuu(Uhd)UfcAfGfUfau A- 758 VPusAfsccaAfgAfAfua GUUUUUUUUCAG 4464 953413.1 1701407. ucuugguaL96 1700899. cuGfaAfaaaaasasc UAUUCUUGGUU 1 1 AD- A- 629 gsusgcu(Ghd)GfaAfUfUfug A- 759 VPusUfsgaaUfaUfCfaa CGGUGCUGGAAU 4465 953294.1 1701169. auauucaaL96 1066884. auUfcCfagcacscsg UUGAUAUUCAU 1 1 AD- A- 630 ususaac(Ahd)UfcAfCfGfuc A- 760 VPusAfsgacAfaAfGfac UAUUAACAUCACG 4466 953421.1 1701423. uuugucuaL96 1070918. guGfaUfguuaasusa UCUUUGUCUC 1 1 AD- A- 631 asgsggg(Chd)AfaAfAfAfcg A- 761 VPusGfscgcUfuUfCfgu AAAGGGGCAAAA 4467 953310.1 1701201. aaagcgcaL96 1700793. uuUfuGfccccususu ACGAAAGCGCA 1 1 AD- A- 632 asasagu(Ghd)AfgUfGfAfcc A- 762 VPusAfsaaaGfcAfGfgu GCAAAGUGAGUG 4468 953296.1 1701173. ugcuuuuaL96 1067146. caCfuCfacuuusgsc ACCUGCUUUUG 1 1 AD- A- 633 asasaaa(Chd)AfcAfGfAfcu A- 763 VPusCfsaacGfcGfAfgu GCAAAAACACAGA 4469 953402.1 1701385. cgcguugaL96 1068540. cuGfuGfuuuuusgsc CUCGCGUUGC 1 1 AD- A- 634 asusuug(Uhd)UfuGfUfAfca A- 764 VPusCfsggaUfcUfUfgu GCAUUUGUUUGU 4470 953312.1 1701205. agauccgaL96 1068458. acAfaAfcaaausgsc ACAAGAUCCGC 1 1 AD- A- 635 ususccc(Chd)AfaAfUfCfac A- 765 VPusAfsuccAfcAfGfug ACUUCCCCAAAUC 4471 953295.1 1701171. uguggauaL96 1700778. auUfuGfgggaasgsu ACUGUGGAUU 1 1 AD- A- 636 asusuaa(Chd)AfuCfAfCfgu A- 766 VPusGfsacaAfaGfAfcg AUAUUAACAUCAC 4472 953420.1 1701421. cuuugucaL96 1070916. ugAfuGfuuaausasu GUCUUUGUCU 1 1 AD- A- 637 csgsucu(Uhd)UfgUfCfUfcu A- 767 VPusCfsugcAfcUfAfga CACGUCUUUGUCU 4473 953423.1 1701427. agugcagaL96 1070936. gaCfaAfagacgsusg CUAGUGCAGU 1 1 AD- A- 638 asasaac(Ahd)CfaGfAfCfuc A- 768 VPusGfscaaCfgCfGfag CAAAAACACAGAC 4474 953403.1 1701387. gcguugcaL96 1068542. ucUfgUfguuuususg UCGCGUUGCA 1 1 AD- A- 639 usgscaa(Ahd)AfaCfAfCfag A- 769 VPusCfsgcgAfgUfCfug CCUGCAAAAACAC 4475 953400.1 1701381. acucgcgaL96 1068534. ugUfuUfuugcasgsg AGACUCGCGU 1 1 AD- A- 640 asascac(Ahd)GfaCfUfCfgc A- 770 VPusUfsugcAfaCfGfcg AAAACACAGACUC 4476 953404.1 1701389. guugcaaaL96 1068546. agUfcUfguguususu GCGUUGCAAG 1 1 AD- A- 641 csusugc(Ahd)GfaUfGfUfga A- 771 VPusCfsggcUfuGfUfca UACUUGCAGAUG 4477 953334.1 1701249. caagccgaL96 1068644. caUfcUfgcaagsusa UGACAAGCCGA 1 1 AD- A- 642 ususuau(Chd)CfgUfAfAfua A- 772 VPusCfscacAfaUfUfau UGUUUAUCCGUA 4478 953418.1 1701417. auuguggaL96 1070894. uaCfgGfauaaascsa AUAAUUGUGGG 1 1 AD- A- 643 gscsaaa(Ahd)AfcAfCfAfga A- 773 VPusAfscgcGfaGfUfcu CUGCAAAAACACA 4479 953401.1 1701383. cucgcguaL96 1068536. guGfuUfuuugcsasg GACUCGCGUU 1 1 AD- A- 644 gsusuua(Uhd)CfcGfUfAfau A- 774 VPusCfsacaAfuUfAfuu CUGUUUAUCCGUA 4480 953417.1 1701415. aauugugaL96 1070892. acGfgAfuaaacsasg AUAAUUGUGG 1 1 AD- A- 645 gsgsgca(Ahd)AfaAfCfGfaa A- 775 VPusUfsugcGfcUfUfuc AGGGGCAAAAAC 4481 953311.1 1701203. agcgcaaaL96 1068252. guUfuUfugcccscsu GAAAGCGCAAG 1 1 AD- A- 646 ususauc(Chd)GfuAfAfUfaa A- 776 VPusCfsccaCfaAfUfua GUUUAUCCGUAA 4482 953419.1 1701419. uugugggaL96 1700905. uuAfcGfgauaasasc UAAUUGUGGGG 1 1

TABLE 3B Exemplary Human VEGF-A siRNA Unmodified Single Strandsand Duplex Sequences SEQ ID Sense SEQ ID mRNA Antisense NO: mRNA Duplex Oligo NO: Target Oligo (Anti- Target Name Name (Sense) Sense Sequence Range Name sense) Antisense Sequence Range AD- A- 777 ACUGAUACAGAACG 1799- A- 907 UAUCGAUCGUUCUGUAUC 1797- 953340. 1701261. AUCGAUA 1819 1068804. AGUCU 1819 1 1 1 AD- A- 778 AAAGACUGAUACAG 1795- A- 908 UAUCGUUCUGUAUCAGUC 1793- 953336. 1701253. AACGAUA 1815 1068796. UUUCC 1815 1 1 1 AD- A- 779 GAGAAAGUGUUUU 2944- A- 909 UCGUAUAUAAAACACUUU 2942- 953363. 1701307. AUAUACGA 2964 1070290. CUCUU 2964 1 1 1 AD- A- 780 AGACUGAUACAGAA 1797- A- 910 UCGAUCGUUCUGUAUCAG 1795- 953338. 1701257. CGAUCGA 1817 1068800. UCUUU 1817 1 1 1 AD- A- 781 CAACUAUUUAUGAG 3061- A- 911 UAUACAUCUCAUAAAUAG 3059- 953367. 1701315. AUGUAUA 3081 1070376. UUGAA 3081 1 1 1 AD- A- 782 AAGACUGAUACAGA 1796- A- 912 UGAUCGUUCUGUAUCAGU 1794- 953337. 1701255. ACGAUCA 1816 1068798. CUUUC 1816 1 1 1 AD- A- 783 AUACAGAACGAUCG 1803- A- 913 UCUGUAUCGAUCGUUCUG 1801- 953342. 1701265. AUACAGA 1823 1068812. UAUCA 1823 1 1 1 AD- A- 784 AACAGUGCUAAUGU 2178- A- 914 UCCAAUAACAUUAGCACU 2176- 953350. 1701281. UAUUGGA 2198 1069342. GUUAA 2198 1 1 1 AD- A- 785 GUGCUAAUGUUAUU 2182- A- 915 UGACACCAAUAACAUUAG 2180- 953352. 1701285. GGUGUCA 2202 1069350. CACUG 2202 1 1 1 AD- A- 786 AACUAUUUAUGAGA 3062- A- 916 UGAUACAUCUCAUAAAUA 3060- 953368. 1701317. UGUAUCA 3082 1070378. GUUGA 3082 1 1 1 AD- A- 787 CAGAACAGUCCUUA 1858- A- 917 UCUGGAUUAAGGACUGUU 1856- 953344. 1701269. AUCCAGA 1878 1068918. CUGUC 1878 1 1 1 AD- A- 788 GACUGAUACAGAAC 1798- A- 918 UUCGAUCGUUCUGUAUCA 1796- 953339. 1701259. GAUCGAA 1818 1068802. GUCUU 1818 1 1 1 AD- A- 789 ACAGCACAACAAAU 1407- A- 919 UAUUCACAUUUGUUGUGC 1405- 953387. 1701355. GUGAAUA 1427 1068170. UGUAG 1427 1 1 1 AD- A- 790 AAAUAGACAUUGCU 3362- A- 920 UCAGAAUAGCAAUGUCUA 3360- 953375. 1701331. AUUCUGA 3382 1070792. UUUUA 3382 1 1 1 AD- A- 791 UAUUGGUGUCUUCA 2192- A- 921 UAUCCAGUGAAGACACCA 2190- 953355. 1701291. CUGGAUA 2212 1069370. AUAAC 2212 1 1 1 AD- A- 792 CUGAUACAGAACGA 1800- A- 922 UUAUCGAUCGUUCUGUAU 1798- 953341. 1701263. UCGAUAA 1820 1068806. CAGUC 1820 1 1 1 AD- A- 793 GCUCUCUUAUUUGU 3096- A- 923 UACCGGUACAAAUAAGAG 3094- 953370. 1701321. ACCGGUA 3116 1070446. AGCAA 3116 1 1 1 AD- A- 794 CACCAUUGAAACCA 2791- A- 924 UAACUAGUGGUUUCAAUG 2789- 953362. 1701305. CUAGUUA 2811 1070096. GUGUG 2811 1 1 1 AD- A- 795 GGCAGCUUGAGUUA 1685- A- 925 UUUCGUUUAACUCAAGCU 1683- 953322. 1701225. AACGAAA 1705 1068596. GCCUC 1705 1 1 1 AD- A- 796 UUAAACGAACGUAC 1696- A- 926 UCUGCAAGUACGUUCGUU 1694- 953332. 1701245. UUGCAGA 1716 1068618. UAACU 1716 1 1 1 AD- A- 797 UCGGUGACAGUCAC 3158- A- 927 UAAGCUAGUGACUGUCAC 3156- 953371. 1701323. UAGCUUA 3178 1070550. CGAUC 3178 1 1 1 AD- A- 798 AGUUAAACGAACGU 1694- A- 928 UGCAAGUACGUUCGUUUA 1692- 953331. 1701243. ACUUGCA 1714 1068614. ACUCA 1714 1 1 1 AD- A- 799 GCAGCUUGAGUUAA 1686- A- 929 UGUUCGUUUAACUCAAGC 1684- 953323. 1701227. ACGAACA 1706 1068598. UGCCU 1706 1 1 1 AD- A- 800 AGUGCUAAUGUUAU 2181- A- 930 UACACCAAUAACAUUAGC 2179- 953351. 1701283. UGGUGUA 2201 1069348. ACUGU 2201 1 1 1 AD- A- 801 CGAAGUGGUGAAGU 1152- A- 931 UCCAUGAACUUCACCACU 1150- 953386. 1701353. UCAUGGA 1172 1067728. UCGUG 1172 1 1 1 AD- A- 802 AAAGCAUUUGUUUG 1611- A- 932 UCUUGUACAAACAAAUGC 1609- 953394. 1701369. UACAAGA 1631 1068448. UUUCU 1631 1 1 1 AD- A- 803 AUGUCCUCACACCA 2782- A- 933 UUUUCAAUGGUGUGAGG 2780- 953359. 1701299. UUGAAAA 2802 1070078. ACAUAG 2802 1 1 1 AD- A- 804 UGAGUUAAACGAAC 1692- A- 934 UAAGUACGUUCGUUUAAC 1690- 953329. 1701239. GUACUUA 1712 1068610. UCAAG 1712 1 1 1 AD- A- 805 ACACCAUUGAAACC 2790- A- 935 UACUAGUGGUUUCAAUGG 2788- 953361. 1701303. ACUAGUA 2810 1070094. UGUGA 2810 1 1 1 AD- A- 806 CAAAAACACAGACU 1656- A- 936 UAACGCGAGUCUGUGUUU 1654- 953319. 1701219. CGCGUUA 1676 1068538. UUGCA 1676 1 1 1 AD- A- 807 UGUCCUCACACCAU 2783- A- 937 UGUUUCAAUGGUGUGAG 2781- 953360. 1701301. UGAAACA 2803 1070080. GACAUA 2803 1 1 1 AD- A- 808 CAGCUUGAGUUAAA 1687- A- 938 UCGUUCGUUUAACUCAAG 1685- 953324. 1701229. CGAACGA 1707 1068600. CUGCC 1707 1 1 1 AD- A- 809 GGUGCUACUGUUUA 3480- A- 939 UUACGGAUAAACAGUAGC 3478- 953378. 1701337. UCCGUAA 3500 1070874. ACCAA 3500 1 1 1 AD- A- 810 UUGCUCUCUUAUUU 3094- A- 940 UCGGUACAAAUAAGAGAG 3092- 953369. 1701319. GUACCGA 3114 1070442. CAAGA 3114 1 1 1 AD- A- 811 UCUUGCUGCUAAAU 2010- A- 941 UUCGGUGAUUUAGCAGCA 2008- 953347. 1701275. CACCGAA 2030 1069188. AGAAA 2030 1 1 1 AD- A- 812 CGGUACUUAUUUAA 2962- A- 942 UGGGAUAUUAAAUAAGU 2960- 953365. 1701311. UAUCCCA 2982 1070326. ACCGUA 2982 1 1 1 AD- A- 813 AAAAUAGACAUUGC 3361- A- 943 UAGAAUAGCAAUGUCUAU 3359- 953374. 1701329. UAUUCUA 3381 1070790. UUUAU 3381 1 1 1 AD- A- 814 ACUUUUCGUCCAAC  657- A- 944 UCCAGAAGUUGGACGAAA  655- 953384. 1701349. UUCUGGA  677 1067266. AGUUU  677 1 1 1 AD- A- 815 UUGGUGCUACUGUU 3478- A- 945 UCGGAUAAACAGUAGCAC 3476- 953376. 1701333. UAUCCGA 3498 1070870. CAAUA 3498 1 1 1 AD- A- 816 GUUAUUGGUGUCUU 2190- A- 946 UCCAGUGAAGACACCAAU 2188- 953354. 1701289. CACUGGA 2210 1069366. AACAU 2210 1 1 1 AD- A- 817 UUCGUCCAACUUCU  661- A- 947 UCAGCCCAGAAGUUGGAC  659- 953385. 1701351. GGGCUGA  681 1067274. GAAAA  681 1 1 1 AD- A- 818 UUCUUGCUGCUAAA 2009- A- 948 UCGGUGAUUUAGCAGCAA 2007- 953346. 1701273. UCACCGA 2029 1069186. GAAAA 2029 1 1 1 AD- A- 819 GGUACUUAUUUAAU 2963- A- 949 UAGGGAUAUUAAAUAAG 2961- 953366. 1701313. AUCCCUA 2983 1070328. UACCGU 2983 1 1 1 AD- A- 820 AUAUUAACAUCACG 3517- A- 950 UCAAAGACGUGAUGUUAA 3515- 953382. 1701345. UCUUUGA 3537 1070912. UAUCU 3537 1 1 1 AD- A- 821 GAGGCAGCUUGAGU 1683- A- 951 UCGUUUAACUCAAGCUGC 1681- 953320. 1701221. UAAACGA 1703 1068592. CUCGC 1703 1 1 1 AD- A- 822 GUGCUACUGUUUAU 3481- A- 952 UUUACGGAUAAACAGUAG 3479- 953379. 1701339. CCGUAAA 3501 1070876. CACCA 3501 1 1 1 AD- A- 823 AGGCAGCUUGAGUU 1684- A- 953 UUCGUUUAACUCAAGCUG 1682- 953321. 1701223. AAACGAA 1704 1068594. CCUCG 1704 1 1 1 AD- A- 824 UGGUGCUACUGUUU 3479- A- 954 UACGGAUAAACAGUAGCA 3477- 953377. 1701335. AUCCGUA 3499 1070872. CCAAU 3499 1 1 1 AD- A- 825 AAGGGGCAAAAACG 1483- A- 955 UCGCUUUCGUUUUUGCCC 1481- 953392. 1701365. AAAGCGA 1503 1700876. CUUUC 1503 1 1 1 AD- A- 826 ACAGUCACUAGCUU 3164- A- 956 UCAAGAUAAGCUAGUGAC 3162- 953373. 1701327. AUCUUGA 3184 1070562. UGUCA 3184 1 1 1 AD- A- 827 ACGGUACUUAUUUA 2961- A- 957 UGGAUAUUAAAUAAGUA 2959- 953364. 1701309. AUAUCCA 2981 1070324. CCGUAU 2981 1 1 1 AD- A- 828 GAGUUAAACGAACG 1693- A- 958 UCAAGUACGUUCGUUUAA 1691- 953330. 1701241. UACUUGA 1713 1068612. CUCAA 1713 1 1 1 AD- A- 829 UGUUAUUGGUGUCU 2189- A- 959 UCAGUGAAGACACCAAUA 2187- 953353. 1701287. UCACUGA 2209 1069364. ACAUU 2209 1 1 1 AD- A- 830 CGACAGAACAGUCC 1855- A- 960 UGAUUAAGGACUGUUCUG 1853- 953343. 1701267. UUAAUCA 1875 1068912. UCGAU 1875 1 1 1 AD- A- 831 AAAAUCAGUUCGAG 1461- A- 961 UCCUUUCCUCGAACUGAU 1459- 953390. 1701361. GAAAGGA 1481 1700873. UUUUU 1481 1 1 1 AD- A- 832 CUUGGAAUUGGAUU 1982- A- 962 UAUGGCGAAUCCAAUUCC 1980- 953345. 1701271. CGCCAUA 2002 1069132. AAGAG 2002 1 1 1 AD- A- 833 AGAGAAGAGACACA 2673- A- 963 UCAACAAUGUGUCUCUUC 2671- 953358. 1701297. UUGUUGA 2693 1069954. UCUUC 2693 1 1 1 AD- A- 834 ACGUCUUUGUCUCU 3528- A- 964 UUGCACUAGAGACAAAGA 3526- 953383. 1701347. AGUGCAA 3548 1070934. CGUGA 3548 1 1 1 AD- A- 835 UGACAGUCACUAGC 3162- A- 965 UAGAUAAGCUAGUGACUG 3160- 953372. 1701325. UUAUCUA 3182 1070558. UCACC 3182 1 1 1 AD- A- 836 UUGAGUUAAACGAA 1691- A- 966 UAGUACGUUCGUUUAACU 1689- 953328. 1701237. CGUACUA 1711 1068608. CAAGC 1711 1 1 1 AD- A- 837 GAAAGCAUUUGUUU 1610- A- 967 UUUGUACAAACAAAUGCU 1608- 953393. 1701367. GUACAAA 1630 1068446. UUCUC 1630 1 1 1 AD- A- 838 AUCACCAUGCAGAU 1342- A- 968 UCGCAUAAUCUGCAUGGU 1340- 953307. 1701195. UAUGCGA 1362 1068040. GAUGU 1362 1 1 1 AD- A- 839 CACCAUGCAGAUUA 1344- A- 969 UUCCGCAUAAUCUGCAUG 1342- 953308. 1701197. UGCGGAA 1364 1068044. GUGAU 1364 1 1 1 AD- A- 840 CUUGAGUUAAACGA 1690- A- 970 UGUACGUUCGUUUAACUC 1688- 953327. 1701235. ACGUACA 1710 1068606. AAGCU 1710 1 1 1 AD- A- 841 UUGCAGAUGUGACA 1710- A- 971 UUCGGCUUGUCACAUCUG 1708- 953335. 1701251. AGCCGAA 1730 1068646. CAAGU 1730 1 1 1 AD- A- 842 ACUAUUUAUGAGAU 3063- A- 972 UAGAUACAUCUCAUAAAU 3061- 953414. 1701409. GUAUCUA 3083 1070380. AGUUG 3083 1 1 1 AD- A- 843 UUUUUUUUCAGUAU 3027- A- 973 UCCAAGAAUACUGAAAAA 3025- 953412. 1701405. UCUUGGA 3047 1700897. AAACC 3047 1 1 1 AD- A- 844 GUUUUAUAUACGGU 2952- A- 974 UAUAAGUACCGUAUAUAA 2950- 953411. 1701403. ACUUAUA 2972 1070306. AACAC 2972 1 1 1 AD- A- 845 GAAAGUGUUUUAU 2946- A- 975 UACCGUAUAUAAAACACU 2944- 953410. 1701401. AUACGGUA 2966 1070294. UUCUC 2966 1 1 1 AD- A- 846 UCACUGGAUGUAUU 2203- A- 976 UCAGUCAAAUACAUCCAG 2201- 953408. 1701397. UGACUGA 2223 1069392. UGAAG 2223 1 1 1 AD- A- 847 GCUUGAGUUAAACG 1689- A- 977 UUACGUUCGUUUAACUCA 1687- 953326. 1701233. AACGUAA 1709 1068604. AGCUG 1709 1 1 1 AD- A- 848 CCUCCGAAACCAUG 1028- A- 978 UAAAGUUCAUGGUUUCGG 1026- 953300. 1701181. AACUUUA 1048 1067482. AGGCC 1048 1 1 1 AD- A- 849 AAAAAUCAGUUCGA 1460- A- 979 UCUUUCCUCGAACUGAUU 1458- 953389. 1701359. GGAAAGA 1480 1700871. UUUUU 1480 1 1 1 AD- A- 850 GAGAAUUCUACAUA 3416- A- 980 UAUUUAGUAUGUAGAAU 3414- 953415. 1701411. CUAAAUA 3436 1070816. UCUCUA 3436 1 1 1 AD- A- 851 AGAUUAUGCGGAUC 1352- A- 981 UAGGUUUGAUCCGCAUAA 1350- 953309. 1701199. AAACCUA 1372 1068060. UCUGC 1372 1 1 1 AD- A- 852 AAAUCAGUUCGAGG 1462- A- 982 UCCCUUUCCUCGAACUGA 1460- 953391. 1701363. AAAGGGA 1482 1068240. UUUUU 1482 1 1 1 AD- A- 853 GCAUUUGUUUGUAC 1614- A- 983 UGAUCUUGUACAAACAAA 1612- 953395. 1701371. AAGAUCA 1634 1068454. UGCUU 1634 1 1 1 AD- A- 854 CACGAAGUGGUGAA 1150- A- 984 UAUGAACUUCACCACUUC 1148- 953303. 1701187. GUUCAUA 1170 1067724. GUGAU 1170 1 1 1 AD- A- 855 UAAUCCAGAAACCU 1870- A- 985 UCAUUUCAGGUUUCUGGA 1868- 953405. 1701391. GAAAUGA 1890 1068942. UUAAG 1890 1 1 1 AD- A- 856 CAUCUUCAAGCCAU 1251- A- 986 UCACAGGAUGGCUUGAAG 1249- 953305. 1701191. CCUGUGA 1271 1067926. AUGUA 1271 1 1 1 AD- A- 857 UGCUACUGUUUAUC 3482- A- 987 UAUUACGGAUAAACAGUA 3480- 953380. 1701341. CGUAAUA 3502 1070878. GCACC 3502 1 1 1 AD- A- 858 UUGCUGCUAAAUCA 2012- A- 988 UGCUCGGUGAUUUAGCAG 2010- 953349. 1701279. CCGAGCA 2032 1069192. CAAGA 2032 1 1 1 AD- A- 859 GAUAUUAACAUCAC 3516- A- 989 UAAAGACGUGAUGUUAA 3514- 953381. 1701343. GUCUUUA 3536 1070910. UAUCUU 3536 1 1 1 AD- A- 860 CUGCAAAAACACAG 1653- A- 990 UGCGAGUCUGUGUUUUUG 1651- 953318. 1701217. ACUCGCA 1673 1068532. CAGGA 1673 1 1 1 AD- A- 861 CUUGCUGCUAAAUC 2011- A- 991 UCUCGGUGAUUUAGCAGC 2009- 953348. 1701277. ACCGAGA 2031 1069190. AAGAA 2031 1 1 1 AD- A- 862 AGAAAGUGUUUUA 2945- A- 992 UCCGUAUAUAAAACACUU 2943- 953409. 1701399. UAUACGGA 2965 1070292. UCUCU 2965 1 1 1 AD- A- 863 AACAUCACCAUGCA 1339- A- 993 UAUAAUCUGCAUGGUGAU 1337- 953306. 1701193. GAUUAUA 1359 1068034. GUUGG 1359 1 1 1 AD- A- 864 CGCAGACGUGUAAA 1634- A- 994 UGGAACAUUUACACGUCU 1632- 953316. 1701213. UGUUCCA 1654 1068494. GCGGA 1654 1 1 1 AD- A- 865 AGCUUGAGUUAAAC 1688- A- 995 UACGUUCGUUUAACUCAA 1686- 953325. 1701231. GAACGUA 1708 1068602. GCUGC 1708 1 1 1 AD- A- 866 GCACUGAAACUUUU  649- A- 996 UUGGACGAAAAGUUUCAG  647- 953299. 1701179. CGUCCAA  669 1067250. UGCGA  669 1 1 1 AD- A- 867 AAUUCUACAUACUA 3419- A- 997 UGAGAUUUAGUAUGUAG 3417- 953416. 1701413. AAUCUCA 3439 1070822. AAUUCU 3439 1 1 1 AD- A- 868 CCGCAGACGUGUAA 1633- A- 998 UGAACAUUUACACGUCUG 1631- 953315. 1701211. AUGUUCA 1653 1068492. CGGAU 1653 1 1 1 AD- A- 869 UCCGCAGACGUGUA 1632- A- 999 UAACAUUUACACGUCUGC 1630- 953314. 1701209. AAUGUUA 1652 1068490. GGAUC 1652 1 1 1 AD- A- 870 CGCACUGAAACUUU  648- A- 1000 UGGACGAAAAGUUUCAGU  646- 953298. 1701177. UCGUCCA  668 1067248. GCGAC  668 1 1 1 AD- A- 871 CCCUCUUGGAAUUG 1978- A- 1001 UCGAAUCCAAUUCCAAGA 1976- 953406. 1701393. GAUUCGA 1998 1069124. GGGAC 1998 1 1 1 AD- A- 872 CAGACGUGUAAAUG 1636- A- 1002 UCAGGAACAUUUACACGU 1634- 953399. 1701379. UUCCUGA 1656 1068498. CUGCG 1656 1 1 1 AD- A- 873 ACGAACGUACUUGC 1700- A- 1003 UACAUCUGCAAGUACGUU 1698- 953333. 1701247. AGAUGUA 1720 1068626. CGUUU 1720 1 1 1 AD- A- 874 AUCCGCAGACGUGU 1631- A- 1004 UACAUUUACACGUCUGCG 1629- 953313. 1701207. AAAUGUA 1651 1068488. GAUCU 1651 1 1 1 AD- A- 875 CAGAAUCAUCACGA 1141- A- 1005 UACCACUUCGUGAUGAUU 1139- 953302. 1701185. AGUGGUA 1161 1067706. CUGCC 1161 1 1 1 AD- A- 876 CCUGCAAAAACACA 1652- A- 1006 UCGAGUCUGUGUUUUUGC 1650- 953317. 1701215. GACUCGA 1672 1068530. AGGAA 1672 1 1 1 AD- A- 877 AGGACAUUGCUGUG 2518- A- 1007 UCCAAAGCACAGCAAUGU 2516- 953357. 1701295. CUUUGGA 2538 1069740. CCUGA 2538 1 1 1 AD- A- 878 GGGCAGAAUCAUCA 1138- A- 1008 UACUUCGUGAUGAUUCUG 1136- 953301. 1701183. CGAAGUA 1158 1067700. CCCUC 1158 1 1 1 AD- A- 879 UGAAGUUCAUGGAU 1160- A- 1009 UAUAGACAUCCAUGAACU 1158- 953304. 1701189. GUCUAUA 1180 1067744. UCACC 1180 1 1 1 AD- A- 880 CGUCGCACUGAAAC  645- A- 1010 UCGAAAAGUUUCAGUGCG  643- 953297. 1701175. UUUUCGA  665 1067242. ACGCC  665 1 1 1 AD- A- 881 GAAAAAAAAUCAGU 1456- A- 1011 UCCUCGAACUGAUUUUUU 1454- 953388. 1701357. UCGAGGA 1476 1700869. UUCUU 1476 1 1 1 AD- A- 882 AUUGGUGUCUUCAC 2193- A- 1012 UCAUCCAGUGAAGACACC 2191- 953407. 1701395. UGGAUGA 2213 1069372. AAUAA 2213 1 1 1 AD- A- 883 AGAUCCGCAGACGU 1629- A- 1013 UAUUUACACGUCUGCGGA 1627- 953397. 1701375. GUAAAUA 1649 1068484. UCUUG 1649 1 1 1 AD- A- 884 GAUCCGCAGACGUG 1630- A- 1014 UCAUUUACACGUCUGCGG 1628- 953398. 1701377. UAAAUGA 1650 1068486. AUCUU 1650 1 1 1 AD- A- 885 UUGUUUGUACAAGA 1618- A- 1015 UUGCGGAUCUUGUACAAA 1616- 953396. 1701373. UCCGCAA 1638 1068462. CAAAU 1638 1 1 1 AD- A- 886 UGCUGUGGACUUGA 2221- A- 1016 UCCCAACUCAAGUCCACA 2219- 953356. 1701293. GUUGGGA 2241 1069428. GCAGU 2241 1 1 1 AD- A- 887 CACGUCUUUGUCUC 3527- A- 1017 UGCACUAGAGACAAAGAC 3525- 953422. 1701425. UAGUGCA 3547 1070932. GUGAU 3547 1 1 1 AD- A- 888 UUUUUUUCAGUAUU 3028- A- 1018 UACCAAGAAUACUGAAAA 3026- 953413. 1701407. CUUGGUA 3048 1700899. AAAAC 3048 1 1 1 AD- A- 889 GUGCUGGAAUUUGA  125- A- 1019 UUGAAUAUCAAAUUCCAG  123- 953294. 1701169. UAUUCAA  145 1066884. CACCG  145 1 1 1 AD- A- 890 UUAACAUCACGUCU 3520- A- 1020 UAGACAAAGACGUGAUGU 3518- 953421. 1701423. UUGUCUA 3540 1070918. UAAUA 3540 1 1 1 AD- A- 891 AGGGGCAAAAACGA 1484- A- 1021 UGCGCUUUCGUUUUUGCC 1482- 953310. 1701201. AAGCGCA 1504 1700793. CCUUU 1504 1 1 1 AD- A- 892 AAAGUGAGUGACCU  415- A- 1022 UAAAAGCAGGUCACUCAC  413- 953296. 1701173. GCUUUUA  435 1067146. UUUGC  435 1 1 1 AD- A- 893 AAAAACACAGACUC 1657- A- 1023 UCAACGCGAGUCUGUGUU 1655- 953402. 1701385. GCGUUGA 1677 1068540. UUUGC 1677 1 1 1 AD- A- 894 AUUUGUUUGUACAA 1616- A- 1024 UCGGAUCUUGUACAAACA 1614- 953312. 1701205. GAUCCGA 1636 1068458. AAUGC 1636 1 1 1 AD- A- 895 UUCCCCAAAUCACU  278- A- 1025 UAUCCACAGUGAUUUGGG  276- 953295. 1701171. GUGGAUA  298 1700778. GAAGU  298 1 1 1 AD- A- 896 AUUAACAUCACGUC 3519- A- 1026 UGACAAAGACGUGAUGUU 3517- 953420. 1701421. UUUGUCA 3539 1070916. AAUAU 3539 1 1 1 AD- A- 897 CGUCUUUGUCUCUA 3529- A- 1027 UCUGCACUAGAGACAAAG 3527- 953423. 1701427. GUGCAGA 3549 1070936. ACGUG 3549 1 1 1 AD- A- 898 AAAACACAGACUCG 1658- A- 1028 UGCAACGCGAGUCUGUGU 1656- 953403. 1701387. CGUUGCA 1678 1068542. UUUUG 1678 1 1 1 AD- A- 899 UGCAAAAACACAGA 1654- A- 1029 UCGCGAGUCUGUGUUUUU 1652- 953400. 1701381. CUCGCGA 1674 1068534. GCAGG 1674 1 1 1 AD- A- 900 AACACAGACUCGCG 1660- A- 1030 UUUGCAACGCGAGUCUGU 1658- 953404. 1701389. UUGCAAA 1680 1068546. GUUUU 1680 1 1 1 AD- A- 901 CUUGCAGAUGUGAC 1709- A- 1031 UCGGCUUGUCACAUCUGC 1707- 953334. 1701249. AAGCCGA 1729 1068644. AAGUA 1729 1 1 1 AD- A- 902 UUUAUCCGUAAUAA 3490- A- 1032 UCCACAAUUAUUACGGAU 3488- 953418. 1701417. UUGUGGA 3510 1070894. AAACA 3510 1 1 1 AD- A- 903 GCAAAAACACAGAC 1655- A- 1033 UACGCGAGUCUGUGUUUU 1653- 953401. 1701383. UCGCGUA 1675 1068536. UGCAG 1675 1 1 1 AD- A- 904 GUUUAUCCGUAAUA 3489- A- 1034 UCACAAUUAUUACGGAUA 3487- 953417. 1701415. AUUGUGA 3509 1070892. AACAG 3509 1 1 1 AD- A- 905 GGGCAAAAACGAAA 1486- A- 1035 UUUGCGCUUUCGUUUUUG 1484- 953311. 1701203. GCGCAAA 1506 1068252. CCCCU 1506 1 1 1 AD- A- 906 UUAUCCGUAAUAAU 3491- A- 1036 UCCCACAAUUAUUACGGA 3489- 953419. 1701419. UGUGGGA 3511 1700905. UAAAC 3511 1 1 1

TABLE 4A Exemplary Human VEGF-A siRNA Modified Single Strandsand Duplex Sequences SEQ ID SEQ ID Sense SEQ ID Antisense NO: NO: Duplex Oligo NO: Oligo (Anti- mRNA target (mRNA Name Name (Sense) Sense Sequence Name sense) Antisense Sequence sequence target) AD- A- 1037 asasaau(Ahd)gad A- 1167 VPusdAsgadAudAgc AUAAAAUAGACAU 4483 953504. 1701069. CadTugcuauucua 1800407.1 aadTgdTcdTauuuusas UGCUAUUCUG 1 1 L96 u AD- A- 1038 asgsugc(Uhd)aad A- 1168 VPusdAscadCcdAau ACAGUGCUAAUGU 4484 953481. 1701023. TgdTuauuggugu 1800384.1 aadCadTudAgcacusg UAUUGGUGUC 1 1 aL96 su AD- A- 1039 asusaca(Ghd)aad A- 1169 VPusdCsugdTadTcga UGAUACAGAACGA 4485 953472. 1701005. CgdAucgauacaga 1800375.1 udCgdTudCuguauscs UCGAUACAGA 1 1 L96 a AD- A- 1040 ascsagc(Ahd)cad A- 1170 VPusdAsuudCadCau CUACAGCACAACA 4486 953517. 1701095. AcdAaaugugaaua 1800420.1 uudGudTgdTgcugusa AAUGUGAAUG 1 1 L96 sg AD- A- 1041 csusgau(Ahd)cad A- 1171 VPusdTsaudCgdAuc GACUGAUACAGAA 4487 953471. 1701003. GadAcgaucgauaa 1800374.1 gudTcdTgdTaucagsu CGAUCGAUAC 1 1 L96 sc AD- A- 1042 gsasgaa(Ahd)gud A- 1172 VPusdCsgudAudAua AAGAGAAAGUGUU 4488 953493. 1701047. GudTuuauauacga 1800396.1 aadAcdAcdTuucucsu UUAUAUACGG 1 1 L96 su AD- A- 1043 asascua(Uhd)uud A- 1173 VPusdGsaudAcdAuc UCAACUAUUUAUG 4489 953498. 1701057. AudGagauguauc 1800401.1 ucdAudAadAuaguus AGAUGUAUCU 1 1 aL96 gsa AD- A- 1044 asasgac(Uhd)gad A- 1174 VPusdGsaudCgdTuc GAAAGACUGAUAC 4490 953467. 1700995. TadCagaacgauca 1800370.1 ugdTadTcdAgucuusu AGAACGAUCG 1 1 L96 sc AD- A- 1045 gsasgaa(Uhd)ucd A- 1175 VPusdAsuudTadGua UAGAGAAUUCUAC 4491 953545. 1701151. TadCauacuaaaua 1800448.1 ugdTadGadAuucucsu AUACUAAAUC 1 1 L96 sa AD- A- 1046 asasaga(Chd)ugd A- 1176 VPusdAsucdGudTcu GGAAAGACUGAUA 4492 953466. 1700993. AudAcagaacgaua 1800369.1 gudAudCadGucuuusc CAGAACGAUC 1 1 L96 sc AD- A- 1047 ascsggu(Ahd)cud A- 1177 VPusdGsgadTadTuaa AUACGGUACUUAU 4493 953494. 1701049. TadTuuaauaucca 1800397.1 adTadAgdTaccgusas UUAAUAUCCC 1 1 L96 u AD- A- 1048 ascsuga(Uhd) acd A- 1178 VPusdAsucdGadTcg AGACUGAUACAGA 4494 953470. 1701001. AgdAacgaucgaua 1800373.1 uudCudGudAucagusc ACGAUCGAUA 1 1 L96 SU AD- A- 1049 csgsaca(Ghd)aad A- 1179 VPusdGsaudTadAgg AUCGACAGAACAG 4495 953473. 1701007. CadGuccuuaauca 1800376.1 acdTgdTudCugucgsa UCCUUAAUCC 1 1 L96 SU AD- A- 1050 csasgaa(Chd)agd A- 1180 VPusdCsugdGadTua GACAGAACAGUCC 4496 953474. 1701009. TcdCuuaauccaga 1800377.1 agdGadCudGuucugsu UUAAUCCAGA 1 1 L96 sc AD- A- 1051 asascag(Uhd)gcd A- 1181 VPusdCscadAudAac UUAACAGUGCUAA 4497 953480. 1701021. TadAuguuauugg 1800383.1 audTadGcdAcuguusa UGUUAUUGGU 1 1 aL96 sa AD- A- 1052 ascsagu(Chd)acd A- 1182 VPusdCsaadGadTaag UGACAGUCACUAG 4498 953503. 1701067. TadGcuuaucuuga 1800406.1 cdTadGudGacuguscs CUUAUCUUGA 1 1 L96 a AD- A- 1053 csusugc(Uhd)gcd A- 1183 VPusdCsucdGgdTga UUCUUGCUGCUAA 4499 953478. 1701017. TadAaucaccgaga 1800381.1 uudTadGcdAgcaagsa AUCACCGAGC 1 1 L96 sa AD- A- 1054 gsasaag(Uhd)gud A- 1184 VPusdAsccdGudAua GAGAAAGUGUUUU 4500 953540. 1701141. TudTauauacggua 1800443.1 uadAadAcdAcuuucsu AUAUACGGUA 1 1 L96 sc AD- A- 1055 gscsucu(Chd)uud A- 1185 VPusdAsccdGgdTaca UUGCUCUCUUAUU 4501 953500. 1701061. AudTuguaccggua 1800403.1 adAudAadGagagcsas UGUACCGGUU 1 1 L96 a AD- A- 1056 ususcuu(Ghd)cud A- 1186 VPusdCsggdTgdAuu UUUUCUUGCUGCU 4502 953476. 1701013. GcdTaaaucaccga 1800379.1 uadGcdAgdCaagaasa AAAUCACCGA 1 1 L96 sa AD- A- 1057 csascca(Uhd)ugd A- 1187 VPusdAsacdTadGug CACACCAUUGAAA 4503 953492. 1701045. AadAccacuaguua 1800395.1 gudTudCadAuggugsu CCACUAGUUC 1 1 L96 sg AD- A- 1058 csgsgua(Chd)uud A- 1188 VPusdGsggdAudAuu UACGGUACUUAUU 4504 953495. 1701051. AudTuaauauccca 1800398.1 aadAudAadGuaccgsu UAAUAUCCCU 1 1 L96 sa AD- A- 1059 csasacu(Ahd)uud A- 1189 VPusdAsuadCadTCUC UUCAACUAUUUAU 4505 953497. 1701055. TadTgagauguaua 1800400.1 adTadAadTaguugsas GAGAUGUAUC 1 1 L96 a AD- A- 1060 usasauc(Chd)agd A- 1190 VPusdCsaudTudCag CUUAAUCCAGAAA 4506 953535. 1701131. AadAccugaaauga 1800438.1 gudTudCudGgauuasa CCUGAAAUGA 1 1 L96 sg AD- A- 1061 asasaua(Ghd)acd A- 1191 VPusdCsagdAadTagc UAAAAUAGACAUU 4507 953505. 1701071. AudTgcuauucuga 1800408.1 adAudGudCuauuusus GCUAUUCUGU 1 1 L96 a AD- A- 1062 asasagc(Ahd)uud A- 1192 VPusdCsuudGudAca AGAAAGCAUUUGU 4508 953524. 1701109. TgdTuuguacaaga 1800427.1 aadCadAadTgcuuusc UUGUACAAGA 1 1 L96 su AD- A- 1063 csusugg(Ahd)aud A- 1193 VPusdAsugdGcdGaa CUCUUGGAAUUGG 4509 953475. 1701011. TgdGauucgccaua 1800378.1 ucdCadAudTccaagsa AUUCGCCAUU 1 1 L96 sg AD- A- 1064 ascsacc(Ahd)uud A- 1194 VPusdAscudAgdTgg UCACACCAUUGAA 4510 953491. 1701043. GadAaccacuagua 1800394.1 uudTcdAadTggugusg ACCACUAGUU 1 1 L96 sa AD- A- 1065 asascau(Chd)acd A- 1195 VPusdAsuadAudCug CCAACAUCACCAU 4511 953436. 1700933. CadTgcagauuaua 1800339.1 cadTgdGudGauguusg GCAGAUUAUG 1 1 L96 sg AD- A- 1066 usgsaca(Ghd)ucd A- 1196 VPusdAsgadTadAgc GGUGACAGUCACU 4512 953502. 1701065. AcdTagcuuaucua 1800405.1 uadGudGadCugucasc AGCUUAUCUU 1 1 L96 sc AD- A- 1067 asgsuua(Ahd)acd A- 1197 VPusdGscadAgdTac UGAGUUAAACGAA 4513 953461. 1700983. GadAcguacuugca 1800364.1 gudTcdGudTuaacusc CGUACUUGCA 1 1 L96 sa AD- A- 1068 ascsuau(Uhd)uad A- 1198 VPusdAsgadTadCauc CAACUAUUUAUGA 4514 953544. 1701149. TgdAgauguaucua 1800447.1 udCadTadAauagusus GAUGUAUCUU 1 1 L96 g AD- A- 1069 ususaaa(Chd)gad A- 1199 VPusdCsugdCadAgu AGUUAAACGAACG 4515 953462. 1700985. AcdGuacuugcaga 1800365.1 acdGudTcdGuuuaasc UACUUGCAGA 1 1 L96 su AD- A- 1070 gsgsuac(Uhd)uad A- 1200 VPusdAsggdGadTau ACGGUACUUAUUU 4516 953496. 1701053. TudTaauaucccua 1800399.1 uadAadTadAguaccsg AAUAUCCCUU 1 1 L96 su AD- A- 1071 csgsaag(Uhd)ggd A- 1201 VPusdCscadTgdAacu CACGAAGUGGUGA 4517 953516. 1701093. TgdAaguucaugga 1800419.1 udCadCcdAcuucgsus AGUUCAUGGA 1 1 L96 g AD- A- 1072 usgsuua(Uhd)ug A- 1202 VPusdCsagdTgdAag AAUGUUAUUGGUG 4518 953483. 1701027. dGudGucuucacu 1800386.1 acdAcdCadAuaacasu UCUUCACUGG 1 1 gaL96 su AD- A- 1073 ususgcu(Chd)ucd A- 1203 VPusdCsggdTadCaaa UCUUGCUCUCUUA 4519 953499. 1701059. TudAuuuguaccga 1800402.1 udAadGadGagcaasgs UUUGUACCGG 1 1 L96 a AD- A- 1074 gsusuuu(Ahd)ua A- 1204 VPusdAsuadAgdTac GUGUUUUAUAUAC 4520 953541. 1701143. dTadCgguacuuau 1800444.1 cgdTadTadTaaaacsas GGUACUUAUU 1 1 aL96 c AD- A- 1075 uscsacu(Ghd)gad A- 1205 VPusdCsagdTcdAaau CUUCACUGGAUGU 4521 953538. 1701137. TgdTauuugacuga 1800441.1 adCadTcdCagugasas AUUUGACUGC 1 1 L96 g AD- A- 1076 cscsucc(Ghd)aad A- 1206 VPusdAsaadGudTca GGCCUCCGAAACC 4522 953430. 1700921. AcdCaugaacuuua 1800333.1 ugdGudTudCggaggsc AUGAACUUUC 1 1 L96 sc AD- A- 1077 usasuug(Ghd)ug A- 1207 VPusdAsucdCadGug GUUAUUGGUGUCU 4523 953485. 1701031. dTcdTucacuggau 1800388.1 aadGadCadCcaauasas UCACUGGAUG 1 1 aL96 c AD- A- 1078 asgsacu(Ghd)aud A- 1208 VPusdCsgadTcdGuu AAAGACUGAUACA 4524 953468. 1700997. AcdAgaacgaucga 1800371.1 cudGudAudCagucusu GAACGAUCGA 1 1 L96 su AD- A- 1079 uscscgc(Ahd)gad A- 1209 VPusdAsacdAudTua GAUCCGCAGACGU 4525 953444. 1700949. CgdTguaaauguua 1800347.1 cadCgdTcdTgcggasu GUAAAUGUUC 1 1 L96 sc AD- A- 1080 gsasguu(Ahd)aad A- 1210 VPusdCsaadGudAcg UUGAGUUAAACGA 4526 953460. 1700981. CgdAacguacuuga 1800363.1 uudCgdTudTaacucsa ACGUACUUGC 1 1 L96 sa AD- A- 1081 asgsaaa(Ghd)ugd A- 1211 VPusdCscgdTadTaua AGAGAAAGUGUUU 4527 953539. 1701139. TudTuauauacgga 1800442.1 adAadCadCuuucuscs UAUAUACGGU 1 1 L96 u AD- A- 1082 gsusuau(Uhd)gg A- 1212 VPusdCscadGudGaa AUGUUAUUGGUGU 4528 953484. 1701029. dTgdTcuucacugg 1800387.1 gadCadCcdAauaacsa CUUCACUGGA 1 1 aL96 su AD- A- 1083 csusuga(Ghd)uud A- 1213 VPusdGsuadCgdTuc AGCUUGAGUUAAA 4529 953457. 1700975. AadAcgaacguaca 1800360.1 gudTudAadCucaagsc CGAACGUACU 1 1 L96 su AD- A- 1084 usgsagu(Uhd)aad A- 1214 VPusdAsagdTadCgu CUUGAGUUAAACG 4530 953459. 1700979. AcdGaacguacuua 1800362.1 ucdGudTudAacucasa AACGUACUUG 1 1 L96 sg AD- A- 1085 asuscac(Chd)aud A- 1215 VPusdCsgcdAudAau ACAUCACCAUGCA 4531 953437. 1700935. GcdAgauuaugcg 1800340.1 cudGcdAudGgugaus GAUUAUGCGG 1 1 aL96 gsu AD- A- 1086 ususgag(Uhd)uad A- 1216 VPusdAsgudAcdGuu GCUUGAGUUAAAC 4532 953458. 1700977. AadCgaacguacua 1800361.1 cgdTudTadAcucaasg GAACGUACUU 1 1 L96 sc AD- A- 1087 gscsagc(Uhd)ugd A- 1217 VPusdGsuudCgdTuu AGGCAGCUUGAGU 4533 953453. 1700967. AgdTuaaacgaaca 1800356.1 aadCudCadAgcugcsc UAAACGAACG 1 1 L96 su AD- A- 1088 csgscac(Uhd)gad A- 1218 VPusdGsgadCgdAaa GUCGCACUGAAAC 4534 953428. 1700917. AadCuuuucgucca 1800331.1 agdTudTcdAgugcgsa UUUUCGUCCA 1 1 L96 sc AD- A- 1089 uscsggu(Ghd)acd A- 1219 VPusdAsagdCudAgu GAUCGGUGACAGU 4535 953501. 1701063. AgdTcacuagcuua 1800404.1 gadCudGudCaccgasu CACUAGCUUA 1 1 L96 sc AD- A- 1090 gsusgcu(Ahd)aud A- 1220 VPusdGsacdAcdCaa CAGUGCUAAUGUU 4536 953482. 1701025. GudTauugguguc 1800385.1 uadAcdAudTagcacsu AUUGGUGUCU 1 1 aL96 sg AD- A- 1091 csgscag(Ahd)cgd A- 1221 VPusdGsgadAcdAuu UCCGCAGACGUGU 4537 953446. 1700953. TgdTaaauguucca 1800349.1 uadCadCgdTcugcgsg AAAUGUUCCU 1 1 L96 sa AD- A- 1092 asgsaga(Ahd)gad A- 1222 VPusdCsaadCadAug GAAGAGAAGAGAC 4538 953488. 1701037. GadCacauuguuga 1800391.1 ugdTcdTcdTucucusu ACAUUGUUGG 1 1 L96 sc AD- A- 1093 usgsaag(Uhd)ucd A- 1223 VPusdAsuadGadCau GGUGAAGUUCAUG 4539 953434. 1700929. AudGgaugucuau 1800337.1 ccdAudGadAcuucasc GAUGUCUAUC 1 1 aL96 sc AD- A- 1094 asasuuc(Uhd)acd A- 1224 VPusdGsagdAudTua AGAAUUCUACAUA 4540 953546. 1701153. AudAcuaaaucuca 1800449.1 gudAudGudAgaauus CUAAAUCUCU 1 1 L96 csu AD- A- 1095 csasgac(Ghd)ugd A- 1225 VPusdCsagdGadAca CGCAGACGUGUAA 4541 953529. 1701119. TadAauguuccuga 1800432.1 uudTadCadCgucugsc AUGUUCCUGC 1 1 L96 sg AD- A- 1096 csascga(Ahd)gud A- 1226 VPusdAsugdAadCuu AUCACGAAGUGGU 4542 953433. 1700927. GgdTgaaguucaua 1800336.1 cadCcdAcdTucgugsa GAAGUUCAUG 1 1 L96 su AD- A- 1097 gscsuug(Ahd)gu A- 1227 VPusdTsacdGudTcgu CAGCUUGAGUUAA 4543 953456. 1700973. dTadAacgaacgua 1800359.1 udTadAcdTcaagcsus ACGAACGUAC 1 1 aL96 g AD- A- 1098 csasucu(Uhd)cad A- 1228 VPusdCsacdAgdGau UACAUCUUCAAGC 4544 953435. 1700931. AgdCcauccuguga 1800338.1 ggdCudTgdAagaugsu CAUCCUGUGU 1 1 L96 sa AD- A- 1099 csascca(Uhd)gcd A- 1229 VPusdTsccdGcdAuaa AUCACCAUGCAGA 4545 953438. 1700937. AgdAuuaugcgga 1800341.1 udCudGcdAuggugsas UUAUGCGGAU 1 1 aL96 u AD- A- 1100 gsgscag(Chd)uud A- 1230 VPusdTsucdGudTuaa GAGGCAGCUUGAG 4546 953452. 1700965. GadGuuaaacgaaa 1800355.1 cdTcdAadGcugccsus UUAAACGAAC 1 1 L96 c AD- A- 1101 asusguc(Chd)ucd A- 1231 VPusdTsuudCadAug CUAUGUCCUCACA 4547 953489. 1701039. AcdAccauugaaaa 1800392.1 gudGudGadGgacausa CCAUUGAAAC 1 1 L96 sg AD- A- 1102 cscsgca(Ghd)acd A- 1232 VPusdGsaadCadTuua AUCCGCAGACGUG 4548 953445. 1700951. GudGuaaauguuc 1800348.1 cdAcdGudCugcggsas UAAAUGUUCC 1 1 aL96 u AD- A- 1103 csasgaa(Uhd)cad A- 1233 VPusdAsccdAcdTuc GGCAGAAUCAUCA 4549 953432. 1700925. TcdAcgaaguggua 1800335.1 gudGadTgdAuucugsc CGAAGUGGUG 1 1 L96 sc AD- A- 1104 gsusgcu(Ahd)cud A- 1234 VPusdTsuadCgdGau UGGUGCUACUGUU 4550 953509. 1701079. GudTuauccguaaa 1800412.1 aadAcdAgdTagcacsc UAUCCGUAAU 1 1 L96 sa AD- A- 1105 usgsucc(Uhd)cad A- 1235 VPusdGsuudTcdAau UAUGUCCUCACAC 4551 953490. 1701041. CadCcauugaaaca 1800393.1 ggdTgdTgdAggacasu CAUUGAAACC 1 1 L96 sa AD- A- 1106 csusgca(Ahd)aad A- 1236 VPusdGscgdAgdTcu UCCUGCAAAAACA 4552 953448. 1700957. AcdAcagacucgca 1800351.1 gudGudTudTugcagsg CAGACUCGCG 1 1 L96 sa AD- A- 1107 gsasggc(Ahd)gcd A- 1237 VPusdCsgudTudAac GCGAGGCAGCUUG 4553 953450. 1700961. TudGaguuaaacga 1800353.1 ucdAadGcdTgccucsg AGUUAAACGA 1 1 L96 sc AD- A- 1108 asusccg(Chd)agd A- 1238 VPusdAscadTudTaca AGAUCCGCAGACG 4554 953443. 1700947. AcdGuguaaaugu 1800346.1 cdGudCudGcggauscs UGUAAAUGUU 1 1 aL96 u AD- A- 1109 gscsauu(Uhd)gud A- 1239 VPusdGsaudCudTgu AAGCAUUUGUUUG 4555 953525. 1701111. TudGuacaagauca 1800428.1 acdAadAcdAaaugcsu UACAAGAUCC 1 1 L96 su AD- A- 1110 gsasaag(Chd)aud A- 1240 VPusdTsugdTadCaaa GAGAAAGCAUUUG 4556 953523. 1701107. TudGuuuguacaaa 1800426.1 cdAadAudGcuuucsus UUUGUACAAG 1 1 L96 c AD- A- 1111 usgsgug(Chd)uad A- 1241 VPusdAscgdGadTaaa AUUGGUGCUACUG 4557 953507. 1701075. CudGuuuauccgu 1800410.1 cdAgdTadGcaccasas UUUAUCCGUA 1 1 aL96 u AD- A- 1112 asgsgca(Ghd)cud A- 1242 VPusdTscgdTudTaac CGAGGCAGCUUGA 4558 953451. 1700963. TgdAguuaaacgaa 1800354.1 udCadAgdCugccuscs GUUAAACGAA 1 1 L96 g AD- A- 1113 gscsacu(Ghd)aad A- 1243 VPusdTsggdAcdGaa UCGCACUGAAACU 4559 953429. 1700919. AcdTuuucguccaa 1800332.1 aadGudTudCagugcsg UUUCGUCCAA 1 1 L96 sa AD- A- 1114 gsascug(Ahd)uad A- 1244 VPusdTscgdAudCgu AAGACUGAUACAG 4560 953469. 1700999. CadGaacgaucgaa 1800372.1 ucdTgdTadTcagucsus AACGAUCGAU 1 1 L96 u AD- A- 1115 ascsgaa(Chd)gud A- 1245 VPusdAscadTcdTgca AAACGAACGUACU 4561 953463. 1700987. AcdTugcagaugua 1800366.1 adGudAcdGuucgusus UGCAGAUGUG 1 1 L96 u AD- A- 1116 csasgcu(Uhd)gad A- 1246 VPusdCsgudTcdGuu GGCAGCUUGAGUU 4562 953454. 1700969. GudTaaacgaacga 1800357.1 uadAcdTcdAagcugsc AAACGAACGU 1 1 L96 sc AD- A- 1117 asgscuu(Ghd)agd A- 1247 VPusdAscgdTudCgu GCAGCUUGAGUUA 4563 953455. 1700971. TudAaacgaacgua 1800358.1 uudAadCudCaagcusg AACGAACGUA 1 1 L96 sc AD- A- 1118 gsasuau(Uhd)aad A- 1248 VPusdAsaadGadCgu AAGAUAUUAACAU 4564 953511. 1701083. CadTcacgucuuua 1800414.1 gadTgdTudAauaucsu CACGUCUUUG 1 1 L96 su AD- A- 1119 cscsugc(Ahd)aad A- 1249 VPusdCsgadGudCug UUCCUGCAAAAAC 4565 953447. 1700955. AadCacagacucga 1800350.1 ugdTudTudTgcaggsa ACAGACUCGC 1 1 L96 sa AD- A- 1120 gsusgcu(Ghd)gad A- 1250 VPusdTsgadAudAuc CGGUGCUGGAAUU 4566 953424. 1700909. AudTugauauucaa 1800327.1 aadAudTcdCagcacscs UGAUAUUCAU 1 1 L96 g AD- A- 1121 ususggu(Ghd)cu A- 1251 VPusdCsggdAudAaa UAUUGGUGCUACU 4567 953506. 1701073. dAcdTguuuauccg 1800409.1 cadGudAgdCaccaasu GUUUAUCCGU 1 1 aL96 sa AD- A- 1122 asusugg(Uhd)gu A- 1252 VPusdCsaudCcdAgu UUAUUGGUGUCUU 4568 953537. 1701135. dCudTcacuggaug 1800440.1 gadAgdAcdAccaausa CACUGGAUGU 1 1 aL96 sa AD- A- 1123 uscsuug(Chd)ugd A- 1253 VPusdTscgdGudGau UUUCUUGCUGCUA 4569 953477. 1701015. CudAaaucaccgaa 1800380.1 uudAgdCadGcaagasa AAUCACCGAG 1 1 L96 sa AD- A- 1124 ususgcu(Ghd)cud A- 1254 VPusdGscudCgdGug UCUUGCUGCUAAA 4570 953479. 1701019. AadAucaccgagca 1800382.1 audTudAgdCagcaasg UCACCGAGCC 1 1 L96 sa AD- A- 1125 asgsauu(Ahd)ugd A- 1255 VPusdAsggdTudTga GCAGAUUAUGCGG 4571 953439. 1700939. CgdGaucaaaccua 1800342.1 ucdCgdCadTaaucusg AUCAAACCUC 1 1 L96 sc AD- A- 1126 gsgsgca(Ghd)aad A- 1256 VPusdAscudTcdGug GAGGGCAGAAUCA 4572 953431. 1700923. TcdAucacgaagua 1800334.1 audGadTudCugcccsu UCACGAAGUG 1 1 L96 sc AD- A- 1127 asusuug(Uhd)uu A- 1257 VPusdCsggdAudCuu GCAUUUGUUUGUA 4573 953442. 1700945. dGudAcaagauccg 1800345.1 gudAcdAadAcaaausg CAAGAUCCGC 1 1 aL96 sc AD- A- 1128 csasaaa(Ahd)cad A- 1258 VPusdAsacdGcdGag UGCAAAAACACAG 4574 953449. 1700959. CadGacucgcguua 1800352.1 ucdTgdTgdTuuuugsc ACUCGCGUUG 1 1 L96 sa AD- A- 1129 usgscua(Chd)ugd A- 1259 VPusdAsuudAcdGga GGUGCUACUGUUU 4575 953510. 1701081. TudTauccguaaua 1800413.1 uadAadCadGuagcasc AUCCGUAAUA 1 1 L96 sc AD- A- 1130 ascsuuu(Uhd)cgd A- 1260 VPusdCscadGadAgu AAACUUUUCGUCC 4576 953514. 1701089. TcdCaacuucugga 1800417.1 ugdGadCgdAaaagusu AACUUCUGGG 1 1 L96 su AD- A- 1131 gsgsugc(Uhd)acd A- 1261 VPusdTsacdGgdAua UUGGUGCUACUGU 4577 953508. 1701077. TgdTuuauccguaa 1800411.1 aadCadGudAgcaccsa UUAUCCGUAA 1 1 L96 sa AD- A- 1132 gscsaaa(Ahd)acd A- 1262 VPusdAscgdCgdAgu CUGCAAAAACACA 4578 953531. 1701123. AcdAgacucgcgua 1800434.1 cudGudGudTuuugcsa GACUCGCGUU 1 1 L96 sg AD- A- 1133 csgsucg(Chd)acd A- 1263 VPusdCsgadAadAgu GGCGUCGCACUGA 4579 953427. 1700915. TgdAaacuuuucga 1800330.1 uudCadGudGcgacgsc AACUUUUCGU 1 1 L96 sc AD- A- 1134 asusauu(Ahd)acd A- 1264 VPusdCsaadAgdAcg AGAUAUUAACAUC 4580 953512. 1701085. AudCacgucuuug 1800415.1 ugdAudGudTaauausc ACGUCUUUGU 1 1 aL96 su AD- A- 1135 asasaac(Ahd)cad A- 1265 VPusdGscadAcdGcg CAAAAACACAGAC 4581 953533. 1701127. GadCucgcguugca 1800436.1 agdTcdTgdTguuuusu UCGCGUUGCA 1 1 L96 sg AD- A- 1136 csusugc(Ahd)gad A- 1266 VPusdCsggdCudTgu UACUUGCAGAUGU 4582 953464. 1700989. TgdTgacaagccga 1800367.1 cadCadTcdTgcaagsus GACAAGCCGA 1 1 L96 a AD- A- 1137 ususuuu(Uhd)uu A- 1267 VPusdCscadAgdAau GGUUUUUUUUCAG 4583 953542. 1701145. dCadGuauucuug 1800445.1 acdTgdAadAaaaaascs UAUUCUUGGU 1 1 gaL96 c AD- A- 1138 asasagu(Ghd)agd A- 1268 VPusdAsaadAgdCag GCAAAGUGAGUGA 4584 953426. 1700913. TgdAccugcuuuua 1800329.1 gudCadCudCacuuusg CCUGCUUUUG 1 1 L96 sc AD- A- 1139 ususcgu(Chd)cad A- 1269 VPusdCsagdCcdCaga UUUUCGUCCAACU 4585 953515. 1701091. AcdTucugggcuga 1800418.1 adGudTgdGacgaasas UCUGGGCUGU 1 1 L96 a AD- A- 1140 asgsgac(Ahd)uud A- 1270 VPusdCscadAadGcac UCAGGACAUUGCU 4586 953487. 1701035. GcdTgugcuuugg 1800390.1 adGcdAadTguccusgs GUGCUUUGGG 1 1 aL96 a AD- A- 1141 asasauc(Ahd)gud A- 1271 VPusdCsccdTudTccu AAAAAUCAGUUCG 4587 953521. 1701103. TcdGaggaaaggga 1800424.1 cdGadAcdTgauuusus AGGAAAGGGA 1 1 L96 u AD- A- 1142 ususccc(Chd)aad A- 1272 VPusdAsucdCadCag ACUUCCCCAAAUC 4588 953425. 1700911. AudCacuguggau 1800328.1 ugdAudTudGgggaasg ACUGUGGAUU 1 1 aL96 su AD- A- 1143 cscscuc(Uhd)ugd A- 1273 VPusdCsgadAudCca GUCCCUCUUGGAA 4589 953536. 1701133. GadAuuggauucg 1800439.1 audTcdCadAgagggsa UUGGAUUCGC 1 1 aL96 sc AD- A- 1144 ususgca(Ghd)aud A- 1274 VPusdTscgdGcdTug ACUUGCAGAUGUG 4590 953465. 1700991. GudGacaagccgaa 1800368.1 ucdAcdAudCugcaasg ACAAGCCGAG 1 1 L96 su AD- A- 1145 csascgu(Chd)uud A- 1275 VPusdGscadCudAga AUCACGUCUUUGU 4591 953552. 1701165. TgdTcucuagugca 1800455.1 gadCadAadGacgugsa CUCUAGUGCA 1 1 L96 su AD- A- 1146 gsasucc(Ghd)cad A- 1276 VPusdCsaudTudAcac AAGAUCCGCAGAC 4592 953528. 1701117. GadCguguaaauga 1800431.1 gdTcdTgdCggaucsus GUGUAAAUGU 1 1 L96 u AD- A- 1147 asasaaa(Uhd)cad A- 1277 VPusdCsuudTcdCuc AAAAAAAUCAGUU 4593 953519. 1701099. GudTcgaggaaaga 1800422.1 gadAcdTgdAuuuuusu CGAGGAAAGG 1 1 L96 su AD- A- 1148 usgscug(Uhd)gg A- 1278 VPusdCsccdAadCuca ACUGCUGUGGACU 4594 953486. 1701033. dAcdTugaguugg 1800389.1 adGudCcdAcagcasgs UGAGUUGGGA 1 1 gaL96 u AD- A- 1149 asasggg(Ghd)cad A- 1279 VPusdCsgcdTudTcgu GAAAGGGGCAAAA 4595 953522. 1701105. AadAacgaaagcga 1800425.1 udTudTgdCcccuusus ACGAAAGCGC 1 1 L96 c AD- A- 1150 usgscaa(Ahd)aad A- 1280 VPusdCsgcdGadGuc CCUGCAAAAACAC 4596 953530. 1701121. CadCagacucgcga 1800433.1 ugdTgdTudTuugcasg AGACUCGCGU 1 1 L96 sg AD- A- 1151 ascsguc(Uhd)uud A- 1281 VPusdTsgcdAcdTaga UCACGUCUUUGUC 4597 953513. 1701087. GudCucuagugcaa 1800416.1 gdAcdAadAgacgusgs UCUAGUGCAG 1 1 L96 a AD- A- 1152 gsgsgca(Ahd)aad A- 1282 VPusdTsugdCgdCuu AGGGGCAAAAACG 4598 953441. 1700943. AcdGaaagcgcaaa 1800344.1 ucdGudTudTugcccsc AAAGCGCAAG 1 1 L96 su AD- A- 1153 asgsggg(Chd)aad A- 1283 VPusdGscgdCudTuc AAAGGGGCAAAAA 4599 953440. 1700941. AadAcgaaagcgca 1800343.1 gudTudTudGccccusu CGAAAGCGCA 1 1 L96 su AD- A- 1154 gsasaaa(Ahd)aad A- 1284 VPusdCscudCgdAac AAGAAAAAAAAUC 4600 953518. 1701097. AudCaguucgagg 1800421.1 ugdAudTudTuuuucsu AGUUCGAGGA 1 1 aL96 su AD- A- 1155 asasaau(Chd)agd A- 1285 VPusdCscudTudCcuc AAAAAAUCAGUUC 4601 953520. 1701101. TudCgaggaaagga 1800423.1 gdAadCudGauuuusus GAGGAAAGGG 1 1 L96 u AD- A- 1156 asasaaa(Chd)acd A- 1286 VPusdCsaadCgdCga GCAAAAACACAGA 4602 953532. 1701125. AgdAcucgcguug 1800435.1 gudCudGudGuuuuus CUCGCGUUGC 1 1 aL96 gsc AD- A- 1157 ususuau(Chd)cgd A- 1287 VPusdCscadCadAuu UGUUUAUCCGUAA 4603 953548. 1701157. TadAuaauugugga 1800451.1 audTadCgdGauaaasc UAAUUGUGGG 1 1 L96 sa AD- A- 1158 asgsauc(Chd)gcd A- 1288 VPusdAsuudTadCac CAAGAUCCGCAGA 4604 953527. 1701115. AgdAcguguaaau 1800430.1 gudCudGcdGgaucusu CGUGUAAAUG 1 1 aL96 sg AD- A- 1159 ususaac(Ahd)ucd A- 1289 VPusdAsgadCadAag UAUUAACAUCACG 4605 953551. 1701163. AcdGucuuugucu 1800454.1 acdGudGadTguuaasu UCUUUGUCUC 1 1 aL96 sa AD- A- 1160 csgsucu(Uhd)ugd A- 1290 VPusdCsugdCadCua CACGUCUUUGUCU 4606 953553. 1701167. TcdTcuagugcaga 1800456.1 gadGadCadAagacgsu CUAGUGCAGU 1 1 L96 sg AD- A- 1161 gsusuua(Uhd)ccd A- 1291 VPusdCsacdAadTuau CUGUUUAUCCGUA 4607 953547. 1701155. GudAauaauugug 1800450.1 udAcdGgdAuaaacsas AUAAUUGUGG 1 1 aL96 g AD- A- 1162 ususguu(Uhd)gu A- 1292 VPusdTsgcdGgdAuc AUUUGUUUGUACA 4608 953526. 1701113. dAcdAagauccgca 1800429.1 uudGudAcdAaacaasa AGAUCCGCAG 1 1 aL96 su AD- A- 1163 ususauc(Chd)gud A- 1293 VPusdCsccdAcdAau GUUUAUCCGUAAU 4609 953549. 1701159. AadTaauuguggga 1800452.1 uadTudAcdGgauaasa AAUUGUGGGG 1 1 L96 sc AD- A- 1164 asascac(Ahd)gad A- 1294 VPusdTsugdCadAcg AAAACACAGACUC 4610 953534. 1701129. CudCgcguugcaaa 1800437.1 cgdAgdTcdTguguusu GCGUUGCAAG 1 1 L96 su AD- A- 1165 ususuuu(Uhd)uc A- 1295 VPusdAsccdAadGaa GUUUUUUUUCAGU 4611 953543. 1701147. dAgdTauucuugg 1800446.1 uadCudGadAaaaaasa AUUCUUGGUU 1 1 uaL96 sc AD- A- 1166 asusuaa(Chd)aud A- 1296 VPusdGsacdAadAga AUAUUAACAUCAC 4612 953550. 1701161. CadCgucuuuguc 1800453.1 cgdTgdAudGuuaausa GUCUUUGUCU 1 1 aL96 su

TABLE 4B Exemplary Human VEGF-A siRNA Unmodified Single Strands and Duplex Sequences SEQ ID Sense SEQ ID mRNA Antisense NO: mRNA Duplex Oligo NO: Target Oligo (Anti- Target Name Name (Sense) Sense Sequence Range Name sense) Antisense Sequence Range AD- A- 1297 AAAAUAGACATUGCU 3361- A- 1427 UAGAAUAGCAATGTCTA 3359- 953504. 1701069. AUUCUA 3381 1800407. UUUUAU 3381 1 1 1 AD- A- 1298 AGUGCUAATGTUAUU 2181- A- 1428 UACACCAAUAACATUAG 2179- 953481. 1701023. GGUGUA 2201 1800384. CACUGU 2201 1 1 1 AD- A- 1299 AUACAGAACGAUCGA 1803- A- 1429 UCUGTATCGAUCGTUCU 1801- 953472. 1701005. UACAGA 1823 1800375. GUAUCA 1823 1 1 1 AD- A- 1300 ACAGCACAACAAAUG 1407- A- 1430 UAUUCACAUUUGUTGTG 1405- 953517. 1701095. UGAAUA 1427 1800420. CUGUAG 1427 1 1 1 AD- A- 1301 CUGAUACAGAACGAU 1800- A- 1431 UTAUCGAUCGUTCTGTA 1798- 953471. 1701003. CGAUAA 1820 1800374. UCAGUC 1820 1 1 1 AD- A- 1302 GAGAAAGUGUTUUA 2944- A- 1432 UCGUAUAUAAAACACTU 2942- 953493. 1701047. UAUACGA 2964 1800396. UCUCUU 2964 1 1 1 AD- A- 1303 AACUAUUUAUGAGA 3062- A- 1433 UGAUACAUCUCAUAAA 3060- 953498. 1701057. UGUAUCA 3082 1800401. UAGUUGA 3082 1 1 1 AD- A- 1304 AAGACUGATACAGAA 1796- A- 1434 UGAUCGTUCUGTATCAG 1794- 953467. 1700995. CGAUCA 1816 1800370. UCUUUC 1816 1 1 1 AD- A- 1305 GAGAAUUCTACAUAC 3416- A- 1435 UAUUTAGUAUGTAGAA 3414- 953545. 1701151. UAAAUA 3436 1800448. UUCUCUA 3436 1 1 1 AD- A- 1306 AAAGACUGAUACAG 1795- A- 1436 UAUCGUTCUGUAUCAGU 1793- 953466. 1700993. AACGAUA 1815 1800369. CUUUCC 1815 1 1 1 AD- A- 1307 ACGGUACUTATUUAA 2961- A- 1437 UGGATATUAAATAAGTA 2959- 953494. 1701049. UAUCCA 2981 1800397. CCGUAU 2981 1 1 1 AD- A- 1308 ACUGAUACAGAACGA 1799- A- 1438 UAUCGATCGUUCUGUAU 1797- 953470. 1701001. UCGAUA 1819 1800373. CAGUCU 1819 1 1 1 AD- A- 1309 CGACAGAACAGUCCU 1855- A- 1439 UGAUTAAGGACTGTUCU 1853- 953473. 1701007. UAAUCA 1875 1800376. GUCGAU 1875 1 1 1 AD- A- 1310 CAGAACAGTCCUUAA 1858- A- 1440 UCUGGATUAAGGACUG 1856- 953474. 1701009. UCCAGA 1878 1800377. UUCUGUC 1878 1 1 1 AD- A- 1311 AACAGUGCTAAUGUU 2178- A- 1441 UCCAAUAACAUTAGCAC 2176- 953480. 1701021. AUUGGA 2198 1800383. UGUUAA 2198 1 1 1 AD- A- 1312 ACAGUCACTAGCUUA 3164- A- 1442 UCAAGATAAGCTAGUGA 3162- 953503. 1701067. UCUUGA 3184 1800406. CUGUCA 3184 1 1 1 AD- A- 1313 CUUGCUGCTAAAUCA 2011- A- 1443 UCUCGGTGAUUTAGCAG 2009- 953478. 1701017. CCGAGA 2031 1800381. CAAGAA 2031 1 1 1 AD- A- 1314 GAAAGUGUTUTAUAU 2946- A- 1444 UACCGUAUAUAAAACA 2944- 953540. 1701141. ACGGUA 2966 1800443. CUUUCUC 2966 1 1 1 AD- A- 1315 GCUCUCUUAUTUGUA 3096- A- 1445 UACCGGTACAAAUAAGA 3094- 953500. 1701061. CCGGUA 3116 1800403. GAGCAA 3116 1 1 1 AD- A- 1316 UUCUUGCUGCTAAAU 2009- A- 1446 UCGGTGAUUUAGCAGCA 2007- 953476. 1701013. CACCGA 2029 1800379. AGAAAA 2029 1 1 1 AD- A- 1317 CACCAUUGAAACCAC 2791- A- 1447 UAACTAGUGGUTUCAAU 2789- 953492. 1701045. UAGUUA 2811 1800395. GGUGUG 2811 1 1 1 AD- A- 1318 CGGUACUUAUTUAAU 2962- A- 1448 UGGGAUAUUAAAUAAG 2960- 953495. 1701051. AUCCCA 2982 1800398. UACCGUA 2982 1 1 1 AD- A- 1319 CAACUAUUTATGAGA 3061- A- 1449 UAUACATCUCATAAATA 3059- 953497. 1701055. UGUAUA 3081 1800400. GUUGAA 3081 1 1 1 AD- A- 1320 UAAUCCAGAAACCUG 1870- A- 1450 UCAUTUCAGGUTUCUGG 1868- 953535. 1701131. AAAUGA 1890 1800438. AUUAAG 1890 1 1 1 AD- A- 1321 AAAUAGACAUTGCUA 3362- A- 1451 UCAGAATAGCAAUGUCU 3360- 953505. 1701071. UUCUGA 3382 1800408. AUUUUA 3382 1 1 1 AD- A- 1322 AAAGCAUUTGTUUGU 1611- A- 1452 UCUUGUACAAACAAATG 1609- 953524. 1701109. ACAAGA 1631 1800427. CUUUCU 1631 1 1 1 AD- A- 1323 CUUGGAAUTGGAUUC 1982- A- 1453 UAUGGCGAAUCCAAUTC 1980- 953475. 1701011. GCCAUA 2002 1800378. CAAGAG 2002 1 1 1 AD- A- 1324 ACACCAUUGAAACCA 2790- A- 1454 UACUAGTGGUUTCAATG 2788- 953491. 1701043. CUAGUA 2810 1800394. GUGUGA 2810 1 1 1 AD- A- 1325 AACAUCACCATGCAG 1339- A- 1455 UAUAAUCUGCATGGUG 1337- 953436. 1700933. AUUAUA 1359 1800339. AUGUUGG 1359 1 1 1 AD- A- 1326 UGACAGUCACTAGCU 3162- A- 1456 UAGATAAGCUAGUGAC 3160- 953502. 1701065. UAUCUA 3182 1800405. UGUCACC 3182 1 1 1 AD- A- 1327 AGUUAAACGAACGU 1694- A- 1457 UGCAAGTACGUTCGUTU 1692- 953461. 1700983. ACUUGCA 1714 1800364. AACUCA 1714 1 1 1 AD- A- 1328 ACUAUUUATGAGAUG 3063- A- 1458 UAGATACAUCUCATAAA 3061- 953544. 1701149. UAUCUA 3083 1800447. UAGUUG 3083 1 1 1 AD- A- 1329 UUAAACGAACGUACU 1696- A- 1459 UCUGCAAGUACGUTCGU 1694- 953462. 1700985. UGCAGA 1716 1800365. UUAACU 1716 1 1 1 AD- A- 1330 GGUACUUATUTAAUA 2963- A- 1460 UAGGGATAUUAAATAA 2961- 953496. 1701053. UCCCUA 2983 1800399. GUACCGU 2983 1 1 1 AD- A- 1331 CGAAGUGGTGAAGUU 1152- A- 1461 UCCATGAACUUCACCAC 1150- 953516. 1701093. CAUGGA 1172 1800419. UUCGUG 1172 1 1 1 AD- A- 1332 UGUUAUUGGUGUCU 2189- A- 1462 UCAGTGAAGACACCAAU 2187- 953483. 1701027. UCACUGA 2209 1800386. AACAUU 2209 1 1 1 AD- A- 1333 UUGCUCUCTUAUUUG 3094- A- 1463 UCGGTACAAAUAAGAG 3092- 953499. 1701059. UACCGA 3114 1800402. AGCAAGA 3114 1 1 1 AD- A- 1334 GUUUUAUATACGGUA 2952- A- 1464 UAUAAGTACCGTATATA 2950- 953541. 1701143. CUUAUA 2972 1800444. AAACAC 2972 1 1 1 AD- A- 1335 UCACUGGATGTAUUU 2203- A- 1465 UCAGTCAAAUACATCCA 2201- 953538. 1701137. GACUGA 2223 1800441. GUGAAG 2223 1 1 1 AD- A- 1336 CCUCCGAAACCAUGA 1028- A- 1466 UAAAGUTCAUGGUTUCG 1026- 953430. 1700921. ACUUUA 1048 1800333. GAGGCC 1048 1 1 1 AD- A- 1337 UAUUGGUGTCTUCAC 2192- A- 1467 UAUCCAGUGAAGACACC 2190- 953485. 1701031. UGGAUA 2212 1800388. AAUAAC 2212 1 1 1 AD- A- 1338 AGACUGAUACAGAAC 1797- A- 1468 UCGATCGUUCUGUAUCA 1795- 953468. 1700997. GAUCGA 1817 1800371. GUCUUU 1817 1 1 1 AD- A- 1339 UCCGCAGACGTGUAA 1632- A- 1469 UAACAUTUACACGTCTG 1630- 953444. 1700949. AUGUUA 1652 1800347. CGGAUC 1652 1 1 1 AD- A- 1340 GAGUUAAACGAACG 1693- A- 1470 UCAAGUACGUUCGTUTA 1691- 953460. 1700981. UACUUGA 1713 1800363. ACUCAA 1713 1 1 1 AD- A- 1341 AGAAAGUGTUTUAUA 2945- A- 1471 UCCGTATAUAAAACACU 2943- 953539. 1701139. UACGGA 2965 1800442. UUCUCU 2965 1 1 1 AD- A- 1342 GUUAUUGGTGTCUUC 2190- A- 1472 UCCAGUGAAGACACCAA 2188- 953484. 1701029. ACUGGA 2210 1800387. UAACAU 2210 1 1 1 AD- A- 1343 CUUGAGUUAAACGA 1690- A- 1473 UGUACGTUCGUTUAACU 1688- 953457. 1700975. ACGUACA 1710 1800360. CAAGCU 1710 1 1 1 AD- A- 1344 UGAGUUAAACGAAC 1692- A- 1474 UAAGTACGUUCGUTUAA 1690- 953459. 1700979. GUACUUA 1712 1800362. CUCAAG 1712 1 1 1 AD- A- 1345 AUCACCAUGCAGAUU 1342- A- 1475 UCGCAUAAUCUGCAUG 1340- 953437. 1700935. AUGCGA 1362 1800340. GUGAUGU 1362 1 1 1 AD- A- 1346 UUGAGUUAAACGAA 1691- A- 1476 UAGUACGUUCGTUTAAC 1689- 953458. 1700977. CGUACUA 1711 1800361. UCAAGC 1711 1 1 1 AD- A- 1347 GCAGCUUGAGTUAAA 1686- A- 1477 UGUUCGTUUAACUCAAG 1684- 953453. 1700967. CGAACA 1706 1800356. CUGCCU 1706 1 1 1 AD- A- 1348 CGCACUGAAACUUUU  648- A- 1478 UGGACGAAAAGTUTCAG  646- 953428. 1700917. CGUCCA  668 1800331. UGCGAC  668 1 1 1 AD- A- 1349 UCGGUGACAGTCACU 3158- A- 1479 UAAGCUAGUGACUGUC 3156- 953501. 1701063. AGCUUA 3178 1800404. ACCGAUC 3178 1 1 1 AD- A- 1350 GUGCUAAUGUTAUUG 2182- A- 1480 UGACACCAAUAACAUTA 2180- 953482. 1701025. GUGUCA 2202 1800385. GCACUG 2202 1 1 1 AD- A- 1351 CGCAGACGTGTAAAU 1634- A- 1481 UGGAACAUUUACACGTC 1632- 953446. 1700953. GUUCCA 1654 1800349. UGCGGA 1654 1 1 1 AD- A- 1352 AGAGAAGAGACACA 2673- A- 1482 UCAACAAUGUGTCTCTU 2671- 953488. 1701037. UUGUUGA 2693 1800391. CUCUUC 2693 1 1 1 AD- A- 1353 UGAAGUUCAUGGAU 1160- A- 1483 UAUAGACAUCCAUGAA 1158- 953434. 1700929. GUCUAUA 1180 1800337. CUUCACC 1180 1 1 1 AD- A- 1354 AAUUCUACAUACUAA 3419- A- 1484 UGAGAUTUAGUAUGUA 3417- 953546. 1701153. AUCUCA 3439 1800449. GAAUUCU 3439 1 1 1 AD- A- 1355 CAGACGUGTAAAUGU 1636- A- 1485 UCAGGAACAUUTACACG 1634- 953529. 1701119. UCCUGA 1656 1800432. UCUGCG 1656 1 1 1 AD- A- 1356 CACGAAGUGGTGAAG 1150- A- 1486 UAUGAACUUCACCACTU 1148- 953433. 1700927. UUCAUA 1170 1800336. CGUGAU 1170 1 1 1 AD- A- 1357 GCUUGAGUTAAACGA 1689- A- 1487 UTACGUTCGUUTAACTC 1687- 953456. 1700973. ACGUAA 1709 1800359. AAGCUG 1709 1 1 1 AD- A- 1358 CAUCUUCAAGCCAUC 1251- A- 1488 UCACAGGAUGGCUTGAA 1249- 953435. 1700931. CUGUGA 1271 1800338. GAUGUA 1271 1 1 1 AD- A- 1359 CACCAUGCAGAUUAU 1344- A- 1489 UTCCGCAUAAUCUGCAU 1342- 953438. 1700937. GCGGAA 1364 1800341. GGUGAU 1364 1 1 1 AD- A- 1360 GGCAGCUUGAGUUA 1685- A- 1490 UTUCGUTUAACTCAAGC 1683- 953452. 1700965. AACGAAA 1705 1800355. UGCCUC 1705 1 1 1 AD- A- 1361 AUGUCCUCACACCAU 2782- A- 1491 UTUUCAAUGGUGUGAG 2780- 953489. 1701039. UGAAAA 2802 1800392. GACAUAG 2802 1 1 1 AD- A- 1362 CCGCAGACGUGUAAA 1633- A- 1492 UGAACATUUACACGUCU 1631- 953445. 1700951. UGUUCA 1653 1800348. GCGGAU 1653 1 1 1 AD- A- 1363 CAGAAUCATCACGAA 1141- A- 1493 UACCACTUCGUGATGAU 1139- 953432. 1700925. GUGGUA 1161 1800335. UCUGCC 1161 1 1 1 AD- A- 1364 GUGCUACUGUTUAUC 3481- A- 1494 UTUACGGAUAAACAGTA 3479- 953509. 1701079. CGUAAA 3501 1800412. GCACCA 3501 1 1 1 AD- A- 1365 UGUCCUCACACCAUU 2783- A- 1495 UGUUTCAAUGGTGTGAG 2781- 953490. 1701041. GAAACA 2803 1800393. GACAUA 2803 1 1 1 AD- A- 1366 CUGCAAAAACACAGA 1653- A- 1496 UGCGAGTCUGUGUTUTU 1651- 953448. 1700957. CUCGCA 1673 1800351. GCAGGA 1673 1 1 1 AD- A- 1367 GAGGCAGCTUGAGUU 1683- A- 1497 UCGUTUAACUCAAGCTG 1681- 953450. 1700961. AAACGA 1703 1800353. CCUCGC 1703 1 1 1 AD- A- 1368 AUCCGCAGACGUGUA 1631- A- 1498 UACATUTACACGUCUGC 1629- 953443. 1700947. AAUGUA 1651 1800346. GGAUCU 1651 1 1 1 AD- A- 1369 GCAUUUGUTUGUACA 1614- A- 1499 UGAUCUTGUACAAACAA 1612- 953525. 1701111. AGAUCA 1634 1800428. AUGCUU 1634 1 1 1 AD- A- 1370 GAAAGCAUTUGUUUG 1610- A- 1500 UTUGTACAAACAAAUGC 1608- 953523. 1701107. UACAAA 1630 1800426. UUUCUC 1630 1 1 1 AD- A- 1371 UGGUGCUACUGUUU 3479- A- 1501 UACGGATAAACAGTAGC 3477- 953507. 1701075. AUCCGUA 3499 1800410. ACCAAU 3499 1 1 1 AD- A- 1372 AGGCAGCUTGAGUUA 1684- A- 1502 UTCGTUTAACUCAAGCU 1682- 953451. 1700963. AACGAA 1704 1800354. GCCUCG 1704 1 1 1 AD- A- 1373 GCACUGAAACTUUUC  649- A- 1503 UTGGACGAAAAGUTUCA  647- 953429. 1700919. GUCCAA  669 1800332. GUGCGA  669 1 1 1 AD- A- 1374 GACUGAUACAGAACG 1798- A- 1504 UTCGAUCGUUCTGTATC 1796- 953469. 1700999. AUCGAA 1818 1800372. AGUCUU 1818 1 1 1 AD- A- 1375 ACGAACGUACTUGCA 1700- A- 1505 UACATCTGCAAGUACGU 1698- 953463. 1700987. GAUGUA 1720 1800366. UCGUUU 1720 1 1 1 AD- A- 1376 CAGCUUGAGUTAAAC 1687- A- 1506 UCGUTCGUUUAACTCAA 1685- 953454. 1700969. GAACGA 1707 1800357. GCUGCC 1707 1 1 1 AD- A- 1377 AGCUUGAGTUAAACG 1688- A- 1507 UACGTUCGUUUAACUCA 1686- 953455. 1700971. AACGUA 1708 1800358. AGCUGC 1708 1 1 1 AD- A- 1378 GAUAUUAACATCACG 3516- A- 1508 UAAAGACGUGATGTUAA 3514- 953511. 1701083. UCUUUA 3536 1800414. UAUCUU 3536 1 1 1 AD- A- 1379 CCUGCAAAAACACAG 1652- A- 1509 UCGAGUCUGUGTUTUTG 1650- 953447. 1700955. ACUCGA 1672 1800350. CAGGAA 1672 1 1 1 AD- A- 1380 GUGCUGGAAUTUGAU  125- A- 1510 UTGAAUAUCAAAUTCCA  123- 953424. 1700909. AUUCAA  145 1800327. GCACCG  145 1 1 1 AD- A- 1381 UUGGUGCUACTGUUU 3478- A- 1511 UCGGAUAAACAGUAGC 3476- 953506. 1701073. AUCCGA 3498 1800409. ACCAAUA 3498 1 1 1 AD- A- 1382 AUUGGUGUCUTCACU 2193- A- 1512 UCAUCCAGUGAAGACAC 2191- 953537. 1701135. GGAUGA 2213 1800440. CAAUAA 2213 1 1 1 AD- A- 1383 UCUUGCUGCUAAAUC 2010- A- 1513 UTCGGUGAUUUAGCAGC 2008- 953477. 1701015. ACCGAA 2030 1800380. AAGAAA 2030 1 1 1 AD- A- 1384 UUGCUGCUAAAUCAC 2012- A- 1514 UGCUCGGUGAUTUAGCA 2010- 953479. 1701019. CGAGCA 2032 1800382. GCAAGA 2032 1 1 1 AD- A- 1385 AGAUUAUGCGGAUC 1352- A- 1515 UAGGTUTGAUCCGCATA 1350- 953439. 1700939. AAACCUA 1372 1800342. AUCUGC 1372 1 1 1 AD- A- 1386 GGGCAGAATCAUCAC 1138- A- 1516 UACUTCGUGAUGATUCU 1136- 953431. 1700923. GAAGUA 1158 1800334. GCCCUC 1158 1 1 1 AD- A- 1387 AUUUGUUUGUACAA 1616- A- 1517 UCGGAUCUUGUACAAA 1614- 953442. 1700945. GAUCCGA 1636 1800345. CAAAUGC 1636 1 1 1 AD- A- 1388 CAAAAACACAGACUC 1656- A- 1518 UAACGCGAGUCTGTGTU 1654- 953449. 1700959. GCGUUA 1676 1800352. UUUGCA 1676 1 1 1 AD- A- 1389 UGCUACUGTUTAUCC 3482- A- 1519 UAUUACGGAUAAACAG 3480- 953510. 1701081. GUAAUA 3502 1800413. UAGCACC 3502 1 1 1 AD- A- 1390 ACUUUUCGTCCAACU  657- A- 1520 UCCAGAAGUUGGACGA  655- 953514. 1701089. UCUGGA  677 1800417. AAAGUUU  677 1 1 1 AD- A- 1391 GGUGCUACTGTUUAU 3480- A- 1521 UTACGGAUAAACAGUA 3478- 953508. 1701077. CCGUAA 3500 1800411. GCACCAA 3500 1 1 1 AD- A- 1392 GCAAAAACACAGACU 1655- A- 1522 UACGCGAGUCUGUGUTU 1653- 953531. 1701123. CGCGUA 1675 1800434. UUGCAG 1675 1 1 1 AD- A- 1393 CGUCGCACTGAAACU  645- A- 1523 UCGAAAAGUUUCAGUG  643- 953427. 1700915. UUUCGA  665 1800330. CGACGCC  665 1 1 1 AD- A- 1394 AUAUUAACAUCACGU 3517- A- 1524 UCAAAGACGUGAUGUT 3515- 953512. 1701085. CUUUGA 3537 1800415. AAUAUCU 3537 1 1 1 AD- A- 1395 AAAACACAGACUCGC 1658- A- 1525 UGCAACGCGAGTCTGTG 1656- 953533. 1701127. GUUGCA 1678 1800436. UUUUUG 1678 1 1 1 AD- A- 1396 CUUGCAGATGTGACA 1709- A- 1526 UCGGCUTGUCACATCTG 1707- 953464. 1700989. AGCCGA 1729 1800367. CAAGUA 1729 1 1 1 AD- A- 1397 UUUUUUUUCAGUAU 3027- A- 1527 UCCAAGAAUACTGAAAA 3025- 953542. 1701145. UCUUGGA 3047 1800445. AAAACC 3047 1 1 1 AD- A- 1398 AAAGUGAGTGACCUG  415- A- 1528 UAAAAGCAGGUCACUC  413- 953426. 1700913. CUUUUA  435 1800329. ACUUUGC  435 1 1 1 AD- A- 1399 UUCGUCCAACTUCUG  661- A- 1529 UCAGCCCAGAAGUTGGA  659- 953515. 1701091. GGCUGA  681 1800418. CGAAAA  681 1 1 1 AD- A- 1400 AGGACAUUGCTGUGC 2518- A- 1530 UCCAAAGCACAGCAATG 2516- 953487. 1701035. UUUGGA 2538 1800390. UCCUGA 2538 1 1 1 AD- A- 1401 AAAUCAGUTCGAGGA 1462- A- 1531 UCCCTUTCCUCGAACTG 1460- 953521. 1701103. AAGGGA 1482 1800424. AUUUUU 1482 1 1 1 AD- A- 1402 UUCCCCAAAUCACUG  278- A- 1532 UAUCCACAGUGAUTUGG  276- 953425. 1700911. UGGAUA  298 1800328. GGAAGU  298 1 1 1 AD- A- 1403 CCCUCUUGGAAUUGG 1978- A- 1533 UCGAAUCCAAUTCCAAG 1976- 953536. 1701133. AUUCGA 1998 1800439. AGGGAC 1998 1 1 1 AD- A- 1404 UUGCAGAUGUGACA 1710- A- 1534 UTCGGCTUGUCACAUCU 1708- 953465. 1700991. AGCCGAA 1730 1800368. GCAAGU 1730 1 1 1 AD- A- 1405 CACGUCUUTGTCUCU 3527- A- 1535 UGCACUAGAGACAAAG 3525- 953552. 1701165. AGUGCA 3547 1800455. ACGUGAU 3547 1 1 1 AD- A- 1406 GAUCCGCAGACGUGU 1630- A- 1536 UCAUTUACACGTCTGCG 1628- 953528. 1701117. AAAUGA 1650 1800431. GAUCUU 1650 1 1 1 AD- A- 1407 AAAAAUCAGUTCGAG 1460- A- 1537 UCUUTCCUCGAACTGAU 1458- 953519. 1701099. GAAAGA 1480 1800422. UUUUUU 1480 1 1 1 AD- A- 1408 UGCUGUGGACTUGAG 2221- A- 1538 UCCCAACUCAAGUCCAC 2219- 953486. 1701033. UUGGGA 2241 1800389. AGCAGU 2241 1 1 1 AD- A- 1409 AAGGGGCAAAAACG 1483- A- 1539 UCGCTUTCGUUTUTGCC 1481- 953522. 1701105. AAAGCGA 1503 1800425. CCUUUC 1503 1 1 1 AD- A- 1410 UGCAAAAACACAGAC 1654- A- 1540 UCGCGAGUCUGTGTUTU 1652- 953530. 1701121. UCGCGA 1674 1800433. UGCAGG 1674 1 1 1 AD- A- 1411 ACGUCUUUGUCUCUA 3528- A- 1541 UTGCACTAGAGACAAAG 3526- 953513. 1701087. GUGCAA 3548 1800416. ACGUGA 3548 1 1 1 AD- A- 1412 GGGCAAAAACGAAA 1486- A- 1542 UTUGCGCUUUCGUTUTU 1484- 953441. 1700943. GCGCAAA 1506 1800344. GCCCCU 1506 1 1 1 AD- A- 1413 AGGGGCAAAAACGA 1484- A- 1543 UGCGCUTUCGUTUTUGC 1482- 953440. 1700941. AAGCGCA 1504 1800343. CCCUUU 1504 1 1 1 AD- A- 1414 GAAAAAAAAUCAGU 1456- A- 1544 UCCUCGAACUGAUTUTU 1454- 953518. 1701097. UCGAGGA 1476 1800421. UUUCUU 1476 1 1 1 AD- A- 1415 AAAAUCAGTUCGAGG 1461- A- 1545 UCCUTUCCUCGAACUGA 1459- 953520. 1701101. AAAGGA 1481 1800423. UUUUUU 1481 1 1 1 AD- A- 1416 AAAAACACAGACUCG 1657- A- 1546 UCAACGCGAGUCUGUG 1655- 953532. 1701125. CGUUGA 1677 1800435. UUUUUGC 1677 1 1 1 AD- A- 1417 UUUAUCCGTAAUAAU 3490- A- 1547 UCCACAAUUAUTACGGA 3488- 953548. 1701157. UGUGGA 3510 1800451. UAAACA 3510 1 1 1 AD- A- 1418 AGAUCCGCAGACGUG 1629- A- 1548 UAUUTACACGUCUGCGG 1627- 953527. 1701115. UAAAUA 1649 1800430. AUCUUG 1649 1 1 1 AD- A- 1419 UUAACAUCACGUCUU 3520- A- 1549 UAGACAAAGACGUGAT 3518- 953551. 1701163. UGUCUA 3540 1800454. GUUAAUA 3540 1 1 1 AD- A- 1420 CGUCUUUGTCTCUAG 3529- A- 1550 UCUGCACUAGAGACAA 3527- 953553. 1701167. UGCAGA 3549 1800456. AGACGUG 3549 1 1 1 AD- A- 1421 GUUUAUCCGUAAUA 3489- A- 1551 UCACAATUAUUACGGAU 3487- 953547. 1701155. AUUGUGA 3509 1800450. AAACAG 3509 1 1 1 AD- A- 1422 UUGUUUGUACAAGA 1618- A- 1552 UTGCGGAUCUUGUACAA 1616- 953526. 1701113. UCCGCAA 1638 1800429. ACAAAU 1638 1 1 1 AD- A- 1423 UUAUCCGUAATAAUU 3491- A- 1553 UCCCACAAUUATUACGG 3489- 953549. 1701159. GUGGGA 3511 1800452. AUAAAC 3511 1 1 1 AD- A- 1424 AACACAGACUCGCGU 1660- A- 1554 UTUGCAACGCGAGTCTG 1658- 953534. 1701129. UGCAAA 1680 1800437. UGUUUU 1680 1 1 1 AD- A- 1425 UUUUUUUCAGTAUUC 3028- A- 1555 UACCAAGAAUACUGAA 3026- 953543. 1701147. UUGGUA 3048 1800446. AAAAAAC 3048 1 1 1 AD- A- 1426 AUUAACAUCACGUCU 3519- A- 1556 UGACAAAGACGTGAUG 3517- 953550. 1701161. UUGUCA 3539 1800453. UUAAUAU 3539 1 1 1

TABLE 5A Exemplary Rat VEGF-A siRNA Modified Single Strands and Duplex Sequences. The mRNA target sequence refers to the target sequence in rat;  the corresponding human target sequence may differ. SEQ ID Sense SEQ ID Antisense SEQ ID NO: Duplex Oligo NO: Oligo NO: mRNA target (mRNA Name Name (Sense) Sense Sequence Name (Antisense) Antisense Sequence sequence target) AD- A- 1557 csgsgaa(Ahd)CfuUf A- 1644 VPusAfsguuGfgAfCf CACGGAAACUU 4613 579911. 1110768 UfUfcguccaacsusa 1100967.1 gaaaAfgUfuuccgsusg UUCGUCCAACU 1 .1 U AD- A- 1558 gsgsaaa(Chd)UfuUf A- 1645 VPusAfsaguUfgGfAf ACGGAAACUUU 4614 579912. 1110769 UfCfguccaacususa 1100969.1 cgaaAfaGfuuuccsgsu UCGUCCAACUU 1 .1 C AD- A- 1559 csgsaca(Ghd)AfaCfA A- 1646 VPusGfsauuAfaGfGf GUCGACAGAAC 4615 579913. 1110770 fGfuccuuaauscsa 1102172.1 acugUfuCfugucgsasc AGUCCUUAAUC 1 .1 C AD- A- 1560 csusgcu(Ahd)AfuGf A- 1647 VPusGfsacaCfcAfAf CUCUGCUAAUG 4616 579914. 1110771 UfUfauugguguscsa 1102638.1 uaacAfuUfagcagsasg UUAUUGGUGUC 1 .1 U AD- A- 1561 uscscga(Ghd)AfuAf A- 1648 VPusGfsuacUfaCfGf UUUCCGAGAUA 4617 579915. 1110772 UfUfccguaguascsa 1103944.1 gaauAfuCfucggasasa UUCCGUAGUAC 1 .1 A AD- A- 1562 csgsaga(Uhd)AfuUf A- 1649 VPusAfsuguAfcUfAf UCCGAGAUAUU 4618 579916. 1110773 CfCfguaguacasusa 1103888.1 cggaAfuAfucucgsgsa CCGUAGUACAU 1 .1 A AD- A- 1563 gscsacg(Ghd)AfaAf A- 1650 VPusUfsggaCfgAfAf UUGCACGGAAA 4619 579917. 1110774 CfUfuuucguccsasa 1100961.1 aaguUfuCfcgugcsasa CUUUUCGUCCA 1 .1 A AD- A- 1564 csascgg(Ahd)AfaCf A- 1651 VPusUfsuggAfcGfAf UGCACGGAAAC 4620 579918. 1110775 UfUfuucguccasasa 1100963.1 aaagUfuUfccgugscsa UUUUCGUCCAA 1 .1 C AD- A- 1565 gsasgau(Ahd)UfuCf A- 1652 VPusUfsaugUfaCfUf CCGAGAUAUUC 4621 579919. 1110776 CfGfuaguacausasa 1103889.1 acggAfaUfaucucsgsg CGUAGUACAUA 1 .1 u AD- A- 1566 ususguu(Uhd)GfuCf A- 1653 VPusUfsgcgGfaUfCf AUUUGUUUGUC 4622 579921. 1110778 CfAfagauccgcsasa 1101976.1 uuggAfcAfaacaasasu CAAGAUCCGCA 1 .1 G AD- A- 1567 ascsgga(Ahd)AfcUf A- 1654 VPusGfsuugGfaCfGf GCACGGAAACU 4623 579922. 1110779 UfUfucguccaascsa 1100965.1 aaaaGfuUfuccgusgsc UUUCGUCCAAC 1 .1 u AD- A- 1568 asuscau(Ghd)CfgGf A- 1655 VPusUfsgagGfuUfUf AGAUCAUGCGG 4624 579923. 1110780 AfUfcaaaccucsasa 1101742.1 gaucCfgCfaugauscsu AUCAAACCUCA 1 .1 c AD- A- 1569 asusgcg(Ghd)AfuCf A- 1656 VPusUfsgguGfaGfGf UCAUGCGGAUC 4625 579924. 1110781 AfAfaccucaccsasa 1101659.1 uuugAfuCfcgcausgsa AAACCUCACCA 1 .1 A AD- A- 1570 gsasuca(Uhd)GfcGf A- 1657 VPusGfsaggUfuUfGf CAGAUCAUGCG 4626 579925. 1110782 GfAfucaaaccuscsa 1101740.1 auccGfcAfugaucsusg GAUCAAACCUC 1 .1 A AD- A- 1571 ususugu(Uhd)UfgUf A- 1658 VPusGfscggAfuCfUf CAUUUGUUUGU 4627 579926. 1110783 CfCfaagauccgscsa 1101974.1 uggaCfaAfacaaasusg CCAAGAUCCGC 1 .1 A AD- A- 1572 gsasucg(Ghd)UfgAf A- 1659 VPusGfscuaGfuGfAf CAGAUCGGUGA 4628 579927. 1110784 CfAfgucacuagscsa 1103783.1 cuguCfaCfcgaucsusg CAGUCACUAGC 1 .1 U AD- A- 1573 asasgau(Chd)CfgCfA A- 1660 VPusUfsuuaCfaCfGf CCAAGAUCCGC 4629 579929. 1110786 fGfacguguaasasa 1101968.1 ucugCfgGfaucuusgsg AGACGUGUAAA 1 .1 U AD- A- 1574 usgsucc(Ahd)AfgAf A- 1661 VPusAfscguCfuGfCf UUUGUCCAAGA 4630 579930. 1110787 UfCfcgcagacgsusa 1101986.1 ggauCfuUfggacasasa UCCGCAGACGU 1 .1 G AD- A- 1575 asuscac(Ghd)UfcUf A- 1662 VPusUfscuaGfaGfAf ACAUCACGUCU 4631 579931. 1110788 UfUfgucucuagsasa 1103887.1 caaaGfaCfgugausgsu UUGUCUCUAGA 1 .1 G AD- A- 1576 usgsaaa(Chd)CfaUfG A- 1663 VPusGfscagAfaAfGf UCUGAAACCAU 4632 579932. 1110789 fAfacuuucugscsa 1101283.1 uucaUfgGfuuucasgsa GAACUUUCUGC 1 .1 U AD- A- 1577 ususguc(Uhd)CfuAf A- 1664 VPusGfsaaaAfcUfGf CUUUGUCUCUA 4633 579933. 1110790 GfAfgcaguuuuscsa 1103908.1 cucuAfgAfgacaasasg GAGCAGUUUUC 1 .1 c AD- A- 1578 csascuu(Chd)CfaGfA A- 1665 VPusUfsuguCfgUfGf CUCACUUCCAG 4634 579934. 1110791 fAfacacgacasasa 1102962.1 uuucUfgGfaagugsasg AAACACGACAA 1 .1 A AD- A- 1579 usgsaaa(Uhd)CfuGf A- 1666 VPusGfsauuGfgAfAf UAUGAAAUCUG 4635 579935. 1110792 UfGfuuuccaauscsa 1103728.1 acacAfgAfuuucasusa UGUUUCCAAUC 1 .1 u AD- A- 1580 gsasaau(Chd)UfgUf A- 1667 VPusAfsgauUfgGfAf AUGAAAUCUGU 4636 579936. 1110793 GfUfuuccaaucsusa 1103730.1 aacaCfaGfauuucsasu GUUUCCAAUCU 1 .1 C AD- A- 1581 ususugu(Chd)UfcUf A- 1668 VPusAfsaaaCfuGfCf UCUUUGUCUCU 4637 579937. 1110794 AfGfagcaguuususa 1103906.1 ucuaGfaGfacaaasgsa AGAGCAGUUUU 1 .1 C AD- A- 1582 usgsucu(Chd)UfaGf A- 1669 VPusGfsgaaAfaCfUf UUUGUCUCUAG 4638 579938. 1110795 AfGfcaguuuucscsa 1103910.1 gcucUfaGfagacasasa AGCAGUUUUCC 1 .1 G AD- A- 1583 asascug(Uhd)AfuUf A- 1670 VPusAfsagcGfuAfAf UAAACUGUAUU 4639 579939. 1110796 GfUfuuuacgcususa 1100541.1 aacaAfuAfcaguususa GUUUUACGCUU 1 .1 U AD- A- 1584 ascsugu(Ahd)UfuGf A- 1671 VPusAfsaagCfgUfAf AAACUGUAUUG 4640 579940. 1110797 UfUfuuacgcuususa 1100543.1 aaacAfaUfacagususu UUUUACGCUUU 1 .1 A AD- A- 1585 csusgua(Uhd)UfgUf A- 1672 VPusUfsaaaGfcGfUf AACUGUAUUGU 4641 579941. 1110798 UfUfuacgcuuusasa 1100545.1 aaaaCfaAfuacagsusu UUUACGCUUUA 1 .1 A AD- A- 1586 usgsuau(Uhd)GfuUf A- 1673 VPusUfsuaaAfgCfGf ACUGUAUUGUU 4642 579942. 1110799 UfUfacgcuuuasasa 1100547.1 uaaaAfcAfauacasgsu UUACGCUUUAA 1 .1 U AD- A- 1587 asusuga(Ahd)AfcCf A- 1674 VPusAfscagAfaUfUf CCAUUGAAACC 4643 579943. 1110800 AfCfuaauucugsusa 1103504.1 agugGfuUfucaausgsg ACUAAUUCUGU 1 .1 C AD- A- 1588 ascsuua(Uhd)UfuAf A- 1675 VPusAfsaaaGfgGfCf GUACUUAUUUA 4644 579944. 1110801 AfUfagcccuuususa 1103594.1 uauuAfaAfuaagusasc AUAGCCCUUUU 1 .1 U AD- A- 1589 uscsucu(Ahd)GfaGf A- 1676 VPusUfscggAfaAfAf UGUCUCUAGAG 4645 579945. 1110802 CfAfguuuuccgsasa 1103914.1 cugcUfcUfagagascsa CAGUUUUCCGA 1 .1 G AD- A- 1590 cscsgag(Ahd)UfaUf A- 1677 VPusUfsguaCfuAfCf UUCCGAGAUAU 4646 579946. 1110803 UfCfcguaguacsasa 1103946.1 ggaaUfaUfcucggsasa UCCGUAGUACA 1 .1 u AD- A- 1591 uscsugg(Ghd)AfuUf A- 1678 VPusUfsuugAfaUfAf GCUCUGGGAUU 4647 579947. 1110804 UfGfauauucaasasa 1100511.1 ucaaAfuCfccagasgsc UGAUAUUCAAA 1 .1 c AD- A- 1592 ususcac(Uhd)GfgAf A- 1679 VPusAfsgucAfaAfCf UCUUCACUGGA 4648 579948. 1110805 UfAfuguuugacsusa 1102658.1 auauCfcAfgugaasgsa UAUGUUUGACU 1 .1 G AD- A- 1593 gsgscuc(Ahd)CfuUf A- 1680 VPusCfsgugUfuUfCf UUGGCUCACUU 4649 579949. 1110806 CfCfagaaacacsgsa 1102954.1 uggaAfgUfgagccsasa CCAGAAACACG 1 .1 A AD- A- 1594 csusucc(Ahd)GfaAf A- 1681 VPusGfsuuuGfuCfGf CACUUCCAGAA 4650 579950. 1110807 AfCfacgacaaascsa 1102966.1 uguuUfcUfggaagsusg ACACGACAAAC 1 .1 C AD- A- 1595 gsascca(Uhd)UfgAf A- 1682 VPusAfsauuAfgUfGf CAGACCAUUGA 4651 579951. 1110808 AfAfccacuaaususa 1103496.1 guuuCfaAfuggucsusg AACCACUAAUU 1 .1 C AD- A- 1596 asgsuca(Chd)UfaGfC A- 1683 VPusCfsucaGfgAfCf ACAGUCACUAG 4652 579953. 1110810 fUfuguccugasgsa 1103805.1 aagcUfaGfugacusgsu CUUGUCCUGAG 1 .1 A AD- A- 1597 ascscac(Ahd)CfaUfU A- 1684 VPusAfsuuuCfaAfAf CCACCACACAU 4653 579954. 1110811 fCfcuuugaaasusa 1103837.1 ggaaUfgUfguggusgsg UCCUUUGAAAU 1 .1 A AD- A- 1598 ascscgg(Ahd)AfaGf A- 1685 VPusGfsguuAfaUfCf UCACCGGAAAG 4654 579955. 1110812 AfCfcgauuaacscsa 1102128.1 ggucUfuUfccggusgsa ACCGAUUAACC 1 .1 A AD- A- 1599 asgsacc(Ghd)AfuUf A- 1686 VPusGfsugaCfaUfGf AAAGACCGAUU 4655 579956. 1110813 AfAfccaugucascsa 1102142.1 guuaAfuCfggucususu AACCAUGUCAC 1 .1 C AD- A- 1600 csusuca(Chd)UfgGf A- 1687 VPusGfsucaAfaCfAf GUCUUCACUGG 4656 579957. 1110814 AfUfauguuugascsa 1102656.1 uaucCfaGfugaagsasc AUAUGUUUGAC 1 .1 U AD- A- 1601 ususggc(Uhd)CfaCf A- 1688 VPusUfsguuUfcUfGf CGUUGGCUCAC 4657 579958. 1110815 UfUfccagaaacsasa 1102950.1 gaagUfgAfgccaascsg UUCCAGAAACA 1 .1 c AD- A- 1602 csuscac(Uhd)UfcCfA A- 1689 VPusGfsucgUfgUfUf GGCUCACUUCC 4658 579959. 1110816 fGfaaacacgascsa 1102958.1 ucugGfaAfgugagscsc AGAAACACGAC 1 .1 A AD- A- 1603 gsusgac(Ahd)GfuCf A- 1690 VPusGfsacaAfgCfUf CGGUGACAGUC 4659 579960. 1110817 AfCfuagcuuguscsa 1103795.1 agugAfcUfgucacscsg ACUAGCUUGUC 1 .1 c AD- A- 1604 gsuscuc(Uhd)AfgAf A- 1691 VPusCfsggaAfaAfCf UUGUCUCUAGA 4660 579961. 1110818 GfCfaguuuuccsgsa 1103912.1 ugcuCfuAfgagacsasa GCAGUUUUCCG 1 .1 A AD- A- 1605 csuscua(Ghd)AfgCf A- 1692 VPusCfsucgGfaAfAf GUCUCUAGAGC 4661 579962. 1110819 AfGfuuuuccgasgsa 1103916.1 acugCfuCfuagagsasc AGUUUUCCGAG 1 .1 A AD- A- 1606 gsusuuu(Chd)CfgAf A- 1693 VPusUfsacgGfaAfUf CAGUUUUCCGA 4662 579963. 1110820 GfAfuauuccgusasa 1103936.1 aucuCfgGfaaaacsusg GAUAUUCCGUA 1 .1 G AD- A- 1607 ususccg(Ahd)GfaUf A- 1694 VPusUfsacuAfcGfGf UUUUCCGAGAU 4663 579964. 1110821 AfUfuccguagusasa 1103942.1 aauaUfcUfcggaasasa AUUCCGUAGUA 1 .1 C AD- A- 1608 ususaaa(Chd)UfgUf A- 1695 VPusCfsguaAfaAfCf UCUUAAACUGU 4664 579965. 1110822 AfUfuguuuuacsgsa 1100535.1 aauaCfaGfuuuaasgsa AUUGUUUUACG 1 .1 C AD- A- 1609 asasacu(Ghd)UfaUf A- 1696 VPusAfsgcgUfaAfAf UUAAACUGUAU 4665 579966. 1110823 UfGfuuuuacgcsusa 1100539.1 acaaUfaCfaguuusasa UGUUUUACGCU 1 .1 U AD- A- 1610 gsasuuc(Ghd)CfcAf A- 1697 VPusAfsuauAfaGfAf UGGAUUCGCCA 4666 579967. 1110824 UfUfuucuuauasusa 1102468.1 aaauGfgCfgaaucscsa UUUUCUUAUAU 1 .1 U AD- A- 1611 uscsacu(Ghd)GfaUf A- 1698 VPusCfsaguCfaAfAf CUUCACUGGAU 4667 579968. 1110825 AfUfguuugacusgsa 1102660.1 cauaUfcCfagugasasg AUGUUUGACUG 1 .1 c AD- A- 1612 gsusugg(Chd)UfcAf A- 1699 VPusGfsuuuCfuGfGf ACGUUGGCUCA 4668 579969. 1110826 CfUfuccagaaascsa 1102948.1 aaguGfaGfccaacsgsu CUUCCAGAAAC 1 .1 A AD- A- 1613 ascsuuc(Chd)AfgAf A- 1700 VPusUfsuugUfcGfUf UCACUUCCAGA 4669 579970. 1110827 AfAfcacgacaasasa 1102964.1 guuuCfuGfgaagusgsa AACACGACAAA 1 .1 C AD- A- 1614 usascuu(Ahd)UfuUf A- 1701 VPusAfsaagGfgCfUf GGUACUUAUUU 4670 579971. 1110828 AfAfuagcccuususa 1103592.1 auuaAfaUfaaguascsc AAUAGCCCUUU 1 .1 U AD- A- 1615 asgsagc(Ahd)GfuUf A- 1702 VPusAfsuauCfuCfGf CUAGAGCAGUU 4671 579972. 1110829 UfUfccgagauasusa 1103924.1 gaaaAfcUfgcucusasg UUCCGAGAUAU 1 .1 U AD- A- 1616 usgsgga(Uhd)UfuGf A- 1703 VPusGfsguuUfgAfAf UCUGGGAUUUG 4672 579973. 1110830 AfUfauucaaacscsa 1100515.1 uaucAfaAfucccasgsa AUAUUCAAACC 1 .1 U AD- A- 1617 gsgsgau(Uhd)UfgAf A- 1704 VPusAfsgguUfuGfAf CUGGGAUUUGA 4673 579974. 1110831 UfAfuucaaaccsusa 1100517.1 auauCfaAfaucccsasg UAUUCAAACCU 1 .1 C AD- A- 1618 gsasuuu(Ghd)AfuAf A- 1705 VPusAfsgagGfuUfUf GGGAUUUGAUA 4674 579975. 1110832 UfUfcaaaccucsusa 1100521.1 gaauAfuCfaaaucscsc UUCAAACCUCU 1 .1 U AD- A- 1619 usasaac(Uhd)GfuAf A- 1706 VPusGfscguAfaAfAf CUUAAACUGUA 4675 579976. 1110833 UfUfguuuuacgscsa 1100537.1 caauAfcAfguuuasasg UUGUUUUACGC 1 .1 U AD- A- 1620 uscsacc(Ghd)GfaAf A- 1707 VPusUfsuaaUfcGfGf UCUCACCGGAA 4676 579977. 1110834 AfGfaccgauuasasa 1102124.1 ucuuUfcCfggugasgsa AGACCGAUUAA 1 .1 C AD- A- 1621 csasccg(Ghd)AfaAf A- 1708 VPusGfsuuaAfuCfGf CUCACCGGAAA 4677 579978. 1110835 GfAfccgauuaascsa 1102126.1 gucuUfuCfcggugsasg GACCGAUUAAC 1 .1 C AD- A- 1622 cscsgga(Ahd)AfgAf A- 1709 VPusUfsgguUfaAfUf CACCGGAAAGA 4678 579979. 1110836 CfCfgauuaaccsasa 1102130.1 cgguCfuUfuccggsusg CCGAUUAACCA 1 .1 U AD- A- 1623 gsasacu(Ghd)GfaUf A- 1710 VPusGfsaaaAfuGfGf UGGAACUGGAU 4679 579980. 1110837 UfCfgccauuuuscsa 1102456.1 cgaaUfcCfaguucscsa UCGCCAUUUUC 1 .1 U AD- A- 1624 csascug(Ghd)AfuAf A- 1711 VPusGfscagUfcAfAf UUCACUGGAUA 4680 579981. 1110838 UfGfuuugacugscsa 1102662.1 acauAfuCfcagugsasa UGUUUGACUGC 1 .1 U AD- A- 1625 gsgsacc(Uhd)UfgUf A- 1712 VPusGfsgucUfgAfUf GAGGACCUUGU 4681 579982. 1110839 GfUfgaucagacscsa 1103464.1 cacaCfaAfgguccsusc GUGAUCAGACC 1 .1 A AD- A- 1626 uscsaga(Chd)CfaUfU A- 1713 VPusUfsaguGfgUfUf GAUCAGACCAU 4682 579983. 1110840 fGfaaaccacusasa 1103490.1 ucaaUfgGfucugasusc UGAAACCACUA 1 .1 A AD- A- 1627 csasuug(Ahd)AfaCf A- 1714 VPusCfsagaAfuUfAf ACCAUUGAAAC 4683 579984. 1110841 CfAfcuaauucusgsa 1103502.1 guggUfuUfcaaugsgsu CACUAAUUCUG 1 .1 U AD- A- 1628 csusaga(Ghd)CfaGf A- 1715 VPusAfsucuCfgGfAf CUCUAGAGCAG 4684 579985. 1110842 UfUfuuccgagasusa 1103920.1 aaacUfgCfucuagsasg UUUUCCGAGAU 1 .1 A AD- A- 1629 usasgag(Chd)AfgUf A- 1716 VPusUfsaucUfcGfGf UCUAGAGCAGU 4685 579986. 1110843 UfUfuccgagausasa 1103922.1 aaaaCfuGfcucuasgsa UUUCCGAGAUA 1 .1 U AD- A- 1630 asgsuuu(Uhd)CfcGf A- 1717 VPusAfscggAfaUfAf GCAGUUUUCCG 4686 579987. 1110844 AfGfauauuccgsusa 1103934.1 ucucGfgAfaaacusgsc AGAUAUUCCGU 1 .1 A AD- A- 1631 gscsgga(Uhd)CfaAf A- 1718 VPusUfsuugGfuGfAf AUGCGGAUCAA 4687 579988. 1110845 AfCfcucaccaasasa 1101748.1 gguuUfgAfuccgcsasu ACCUCACCAAA 1 .1 G AD- A- 1632 asgscau(Uhd)UfgUf A- 1719 VPusAfsucuUfgGfAf AAAGCAUUUGU 4688 579989. 1110846 UfUfguccaagasusa 1101962.1 caaaCfaAfaugcususu UUGUCCAAGAU 1 .1 C AD- A- 1633 uscsuca(Chd)CfgGf A- 1720 VPusAfsaucGfgUfCf CCUCUCACCGG 4689 579990. 1110847 AfAfagaccgaususa 1102120.1 uuucCfgGfugagasgsg AAAGACCGAUU 1 .1 A AD- A- 1634 uscsuag(Ahd)GfcAf A- 1721 VPusUfscucGfgAfAf UCUCUAGAGCA 4690 579992. 1110849 GfUfuuuccgagsasa 1103918.1 aacuGfcUfcuagasgsa GUUUUCCGAGA 1 .1 U AD- A- 1635 asusugc(Ahd)CfgGf A- 1722 VPusAfscgaAfaAfGf GGAUUGCACGG 4691 579993. 1110850 AfAfacuuuucgsusa 1100955.1 uuucCfgUfgcaauscsc AAACUUUUCGU 1 .1 C AD- A- 1636 gscsucu(Ghd)GfgAf A- 1723 VPusUfsgaaUfaUfCf GUGCUCUGGGA 4692 579995. 1110852 UfUfugauauucsasa 1100507.1 aaauCfcCfagagcsasc UUUGAUAUUCA 1 .1 A AD- A- 1637 usgsagc(Chd)UfuGf A- 1724 VPusUfsccgCfuCfUf UGUGAGCCUUG 4693 579996. 1110853 UfUfcagagcggsasa 1101930.1 gaacAfaGfgcucascsa UUCAGAGCGGA 1 .1 G AD- A- 1638 asgsccu(Uhd)GfuUf A- 1725 VPusUfscucCfgCfUf UGAGCCUUGUU 4694 579997. 1110854 CfAfgagcggagsasa 1101934.1 cugaAfcAfaggcuscsa CAGAGCGGAGA 1 .1 A AD- A- 1639 csasuuu(Ghd)UfuUf A- 1726 VPusGfsgauCfuUfGf AGCAUUUGUUU 4695 579998. 1110855 GfUfccaagaucscsa 1101966.1 gacaAfaCfaaaugscsu GUCCAAGAUCC 1 .1 G AD- A- 1640 gsgsaaa(Ghd)AfcCf A- 1727 VPusCfsaugGfuUfAf CCGGAAAGACC 4696 579999. 1110856 GfAfuuaaccausgsa 1102134.1 aucgGfuCfuuuccsgsg GAUUAACCAUG 1 .1 U AD- A- 1641 gsasaag(Ahd)CfcGf A- 1728 VPusAfscauGfgUfUf CGGAAAGACCG 4697 580000. 1110857 AfUfuaaccaugsusa 1102136.1 aaucGfgUfcuuucscsg AUUAACCAUGU 1 .1 C AD- A- 1642 asasgac(Chd)GfaUfU A- 1729 VPusUfsgacAfuGfGf GAAAGACCGAU 4698 580001. 1110858 fAfaccaugucsasa 1102140.1 uuaaUfcGfgucuususc UAACCAUGUCA 1 .1 C AD- A- 1643 gsasccg(Ahd)UfuAf A- 1730 VPusGfsgugAfcAfUf AAGACCGAUUA 4699 580002. 1110859 AfCfcaugucacscsa 1102144.1 gguuAfaUfcggucsusu ACCAUGUCACC 1 .1 A

TABLE 5B Exemplary Rat VEGF-A siRNA Unmodified Single Strands and Duplex Sequences Sense SEQ mRNA Antisense SEQ ID mRNA Duplex Oligo ID NO: Target Oligo NO: Antisense Target Name Name (Sense) Sense Sequence Range Name (Antisense) Sequence Range AD- A- 1731 CGGAAACUUUUCGUC  631- A- 1818 UAGUUGGACGAAA  629- 57991 11107 CAACUA  651 1100967.1 AGUUUCCGUG  651 1.1 68.1 AD- A- 1732 GGAAACUUUUCGUCC  632- A- 1819 UAAGUUGGACGAA  630- 57991 11107 AACUUA  652 1100969.1 AAGUUUCCGU  652 2.1 69.1 AD- A- 1733 CGACAGAACAGUCCU 1689- A- 1820 UGAUUAAGGACUG 1687- 57991 11107 UAAUCA 1709 1102172.1 UUCUGUCGAC 1709 3.1 70.1 AD- A- 1734 CUGCUAAUGUUAUUG 2020- A- 1821 UGACACCAAUAAC 2018- 57991 11107 GUGUCA 2040 1102638.1 AUUAGCAGAG 2040 4.1 71.1 AD- A- 1735 UCCGAGAUAUUCCGU 3364- A- 1822 UGUACUACGGAAU 3362- 57991 11107 AGUACA 3384 1103944.1 AUCUCGGAAA 3384 5.1 72.1 AD- A- 1736 CGAGAUAUUCCGUAG 3366- A- 1823 UAUGUACUACGGA 3364- 57991 11107 UACAUA 3386 1103888.1 AUAUCUCGGA 3386 6.1 73.1 AD- A- 1737 GCACGGAAACUUUUC  628- A- 1824 UUGGACGAAAAGU  626- 57991 11107 GUCCAA  648 1100961.1 UUCCGUGCAA  648 7.1 74.1 AD- A- 1738 CACGGAAACUUUUCG  629- A- 1825 UUUGGACGAAAAG  627- 57991 11107 UCCAAA  649 1100963.1 UUUCCGUGCA  649 8.1 75.1 AD- A- 1739 GAGAUAUUCCGUAGU 3367- A- 1826 UUAUGUACUACGG 3365- 57991 11107 ACAUAA 3387 1103889.1 AAUAUCUCGG 3387 9.1 76.1 AD- A- 1740 UUGUUUGUCCAAGAU 1468- A- 1827 UUGCGGAUCUUGG 1466- 57992 11107 CCGCAA 1488 1101976.1 ACAAACAAAU 1488 1.1 78.1 AD- A- 1741 ACGGAAACUUUUCGU  630- A- 1828 UGUUGGACGAAAA  628- 57992 11107 CCAACA  650 1100965.1 GUUUCCGUGC  650 2.1 79.1 AD- A- 1742 AUCAUGCGGAUCAAA 1327- A- 1829 UUGAGGUUUGAUC 1325- 57992 11107 CCUCAA 1347 1101742.1 CGCAUGAUCU 1347 3.1 80.1 AD- A- 1743 AUGCGGAUCAAACCU 1330- A- 1830 UUGGUGAGGUUUG 1328- 57992 11107 CACCAA 1350 1101659.1 AUCCGCAUGA 1350 4.1 81.1 AD- A- 1744 GAUCAUGCGGAUCAA 1326- A- 1831 UGAGGUUUGAUCC 1324- 57992 11107 ACCUCA 1346 1101740.1 GCAUGAUCUG 1346 5.1 82.1 AD- A- 1745 UUUGUUUGUCCAAGA 1467- A- 1832 UGCGGAUCUUGGA 1465- 57992 11107 UCCGCA 1487 1101974.1 CAAACAAAUG 1487 6.1 83.1 AD- A- 1746 GAUCGGUGACAGUCA 2972- A- 1833 UGCUAGUGACUGU 2970- 57992 11107 CUAGCA 2992 1103783.1 CACCGAUCUG 2992 7.1 84.1 AD- A- 1747 AAGAUCCGCAGACGU 1478- A- 1834 UUUUACACGUCUG 1476- 57992 11107 GUAAAA 1498 1101968.1 CGGAUCUUGG 1498 9.1 86.1 AD- A- 1748 UGUCCAAGAUCCGCA 1473- A- 1835 UACGUCUGCGGAU 1471- 57993 11107 GACGUA 1493 1101986.1 CUUGGACAAA 1493 0.1 87.1 AD- A- 1749 AUCACGUCUUUGUCU 3337- A- 1836 UUCUAGAGACAAA 3335- 57993 11107 CUAGAA 3357 1103887.1 GACGUGAUGU 3357 1.1 88.1 AD- A- 1750 UGAAACCAUGAACUU 1008- A- 1837 UGCAGAAAGUUCA 1006- 57993 11107 UCUGCA 1028 1101283.1 UGGUUUCAGA 1028 2.1 89.1 AD- A- 1751 UUGUCUCUAGAGCAG 3346- A- 1838 UGAAAACUGCUCU 3344- 57993 11107 UUUUCA 3366 1103908.1 AGAGACAAAG 3366 3.1 90.1 AD- A- 1752 CACUUCCAGAAACACG 2222- A- 1839 UUUGUCGUGUUUC 2220- 57993 11107 ACAAA 2242 1102962.1 UGGAAGUGAG 2242 4.1 91.1 AD- A- 1753 UGAAAUCUGUGUUUC 2941- A- 1840 UGAUUGGAAACAC 2939- 57993 11107 CAAUCA 2961 1103728.1 AGAUUUCAUA 2961 5.1 92.1 AD- A- 1754 GAAAUCUGUGUUUCC 2942- A- 1841 UAGAUUGGAAACA 2940- 57993 11107 AAUCUA 2962 1103730.1 CAGAUUUCAU 2962 6.1 93.1 AD- A- 1755 UUUGUCUCUAGAGCA 3345- A- 1842 UAAAACUGCUCUA 3343- 57993 11107 GUUUUA 3365 1103906.1 GAGACAAAGA 3365 7.1 94.1 AD- A- 1756 UGUCUCUAGAGCAGU 3347- A- 1843 UGGAAAACUGCUC 3345- 57993 11107 UUUCCA 3367 1103910.1 UAGAGACAAA 3367 8.1 95.1 AD- A- 1757 AACUGUAUUGUUUUA  167- A- 1844 UAAGCGUAAAACA  165- 57993 11107 CGCUUA  187 1100541.1 AUACAGUUUA  187 9.1 96.1 AD- A- 1758 ACUGUAUUGUUUUAC  168- A- 1845 UAAAGCGUAAAAC  166- 57994 11107 GCUUUA  188 1100543.1 AAUACAGUUU  188 0.1 97.1 AD- A- 1759 CUGUAUUGUUUUACG  169- A- 1846 UUAAAGCGUAAAA  167- 57994 11107 CUUUAA  189 1100545.1 CAAUACAGUU  189 1.1 98.1 AD- A- 1760 UGUAUUGUUUUACGC  170- A- 1847 UUUAAAGCGUAAA  168- 57994 11107 UUUAAA  190 1100547.1 ACAAUACAGU  190 2.1 99.1 AD- A- 1761 AUUGAAACCACUAAU 2611- A- 1848 UACAGAAUUAGUG 2609- 57994 11108 UCUGUA 2631 1103504.1 GUUUCAAUGG 2631 3.1 00.1 AD- A- 1762 ACUUAUUUAAUAGCC 2764- A- 1849 UAAAAGGGCUAUU 2762- 57994 11108 CUUUUA 2784 1103594.1 AAAUAAGUAC 2784 4.1 01.1 AD- A- 1763 UCUCUAGAGCAGUUU 3349- A- 1850 UUCGGAAAACUGC 3347- 57994 11108 UCCGAA 3369 1103914.1 UCUAGAGACA 3369 5.1 02.1 AD- A- 1764 CCGAGAUAUUCCGUA 3365- A- 1851 UUGUACUACGGAA 3363- 57994 11108 GUACAA 3385 1103946.1 UAUCUCGGAA 3385 6.1 03.1 AD- A- 1765 UCUGGGAUUUGAUAU  129- A- 1852 UUUUGAAUAUCAA  127- 57994 11108 UCAAAA  149 1100511.1 AUCCCAGAGC  149 7.1 04.1 AD- A- 1766 UUCACUGGAUAUGUU 2040- A- 1853 UAGUCAAACAUAU 2038- 57994 11108 UGACUA 2060 1102658.1 CCAGUGAAGA 2060 8.1 05.1 AD- A- 1767 GGCUCACUUCCAGAA 2218- A- 1854 UCGUGUUUCUGGA 2216- 57994 11108 ACACGA 2238 1102954.1 AGUGAGCCAA 2238 9.1 06.1 AD- A- 1768 CUUCCAGAAACACGAC 2224- A- 1855 UGUUUGUCGUGUU 2222- 57995 11108 AAACA 2244 1102966.1 UCUGGAAGUG 2244 0.1 07.1 AD- A- 1769 GACCAUUGAAACCAC 2607- A- 1856 UAAUUAGUGGUUU 2605- 57995 11108 UAAUUA 2627 1103496.1 CAAUGGUCUG 2627 1.1 08.1 AD- A- 1770 AGUCACUAGCUUGUC 2982- A- 1857 UCUCAGGACAAGC 2980- 57995 11108 CUGAGA 3002 1103805.1 UAGUGACUGU 3002 3.1 10.1 AD- A- 1771 ACCACACAUUCCUUUG 3049- A- 1858 UAUUUCAAAGGAA 3047- 57995 11108 AAAUA 3069 1103837.1 UGUGUGGUGG 3069 4.1 11.1 AD- A- 1772 ACCGGAAAGACCGAU 1639- A- 1859 UGGUUAAUCGGUC 1637- 57995 11108 UAACCA 1659 1102128.1 UUUCCGGUGA 1659 5.1 12.1 AD- A- 1773 AGACCGAUUAACCAU 1646- A- 1860 UGUGACAUGGUUA 1644- 57995 11108 GUCACA 1666 1102142.1 AUCGGUCUUU 1666 6.1 13.1 AD- A- 1774 CUUCACUGGAUAUGU 2039- A- 1861 UGUCAAACAUAUC 2037- 57995 11108 UUGACA 2059 1102656.1 CAGUGAAGAC 2059 7.1 14.1 AD- A- 1775 UUGGCUCACUUCCAG 2216- A- 1862 UUGUUUCUGGAAG 2214- 57995 11108 AAACAA 2236 1102950.1 UGAGCCAACG 2236 8.1 15.1 AD- A- 1776 CUCACUUCCAGAAACA 2220- A- 1863 UGUCGUGUUUCUG 2218- 57995 11108 CGACA 2240 1102958.1 GAAGUGAGCC 2240 9.1 16.1 AD- A- 1777 GUGACAGUCACUAGC 2977- A- 1864 UGACAAGCUAGUG 2975- 57996 11108 UUGUCA 2997 1103795.1 ACUGUCACCG 2997 0.1 17.1 AD- A- 1778 GUCUCUAGAGCAGUU 3348- A- 1865 UCGGAAAACUGCU 3346- 57996 11108 UUCCGA 3368 1103912.1 CUAGAGACAA 3368 1.1 18.1 AD- A- 1779 CUCUAGAGCAGUUUU 3350- A- 1866 UCUCGGAAAACUG 3348- 57996 11108 CCGAGA 3370 1103916.1 CUCUAGAGAC 3370 2.1 19.1 AD- A- 1780 GUUUUCCGAGAUAUU 3360- A- 1867 UUACGGAAUAUCU 3358- 57996 11108 CCGUAA 3380 1103936.1 CGGAAAACUG 3380 3.1 20.1 AD- A- 1781 UUCCGAGAUAUUCCG 3363- A- 1868 UUACUACGGAAUA 3361- 57996 11108 UAGUAA 3383 1103942.1 UCUCGGAAAA 3383 4.1 21.1 AD- A- 1782 UUAAACUGUAUUGUU  164- A- 1869 UCGUAAAACAAUA  162- 57996 11108 UUACGA  184 1100535.1 CAGUUUAAGA  184 5.1 22.1 AD- A- 1783 AAACUGUAUUGUUUU  166- A- 1870 UAGCGUAAAACAA  164- 57996 11108 ACGCUA  186 1100539.1 UACAGUUUAA  186 6.1 23.1 AD- A- 1784 GAUUCGCCAUUUUCU 1830- A- 1871 UAUAUAAGAAAAU 1828- 57996 11108 UAUAUA 1850 1102468.1 GGCGAAUCCA 1850 7.1 24.1 AD- A- 1785 UCACUGGAUAUGUUU 2041- A- 1872 UCAGUCAAACAUA 2039- 57996 11108 GACUGA 2061 1102660.1 UCCAGUGAAG 2061 8.1 25.1 AD- A- 1786 GUUGGCUCACUUCCA 2215- A- 1873 UGUUUCUGGAAGU 2213- 57996 11108 GAAACA 2235 1102948.1 GAGCCAACGU 2235 9.1 26.1 AD- A- 1787 ACUUCCAGAAACACG 2223- A- 1874 UUUUGUCGUGUUU 2221- 57997 11108 ACAAAA 2243 1102964.1 CUGGAAGUGA 2243 0.1 27.1 AD- A- 1788 UACUUAUUUAAUAGC 2763- A- 1875 UAAAGGGCUAUUA 2761- 57997 11108 CCUUUA 2783 1103592.1 AAUAAGUACC 2783 1.1 28.1 AD- A- 1789 AGAGCAGUUUUCCGA 3354- A- 1876 UAUAUCUCGGAAA 3352- 57997 11108 GAUAUA 3374 1103924.1 ACUGCUCUAG 3374 2.1 29.1 AD- A- 1790 UGGGAUUUGAUAUUC  131- A- 1877 UGGUUUGAAUAUC  129- 57997 11108 AAACCA  151 1100515.1 AAAUCCCAGA  151 3.1 30.1 AD- A- 1791 GGGAUUUGAUAUUCA  132- A- 1878 UAGGUUUGAAUAU  130- 57997 11108 AACCUA  152 1100517.1 CAAAUCCCAG  152 4.1 31.1 AD- A- 1792 GAUUUGAUAUUCAAA  134- A- 1879 UAGAGGUUUGAAU  132- 57997 11108 CCUCUA  154 1100521.1 AUCAAAUCCC  154 5.1 32.1 AD- A- 1793 UAAACUGUAUUGUUU  165- A- 1880 UGCGUAAAACAAU  163- 57997 11108 UACGCA  185 1100537.1 ACAGUUUAAG  185 6.1 33.1 AD- A- 1794 UCACCGGAAAGACCG 1637- A- 1881 UUUAAUCGGUCUU 1635- 57997 11108 AUUAAA 1657 1102124.1 UCCGGUGAGA 1657 7.1 34.1 AD- A- 1795 CACCGGAAAGACCGA 1638- A- 1882 UGUUAAUCGGUCU 1636- 57997 11108 UUAACA 1658 1102126.1 UUCCGGUGAG 1658 8.1 35.1 AD- A- 1796 CCGGAAAGACCGAUU 1640- A- 1883 UUGGUUAAUCGGU 1638- 57997 11108 AACCAA 1660 1102130.1 CUUUCCGGUG 1660 9.1 36.1 AD- A- 1797 GAACUGGAUUCGCCA 1824- A- 1884 UGAAAAUGGCGAA 1822- 57998 11108 UUUUCA 1844 1102456.1 UCCAGUUCCA 1844 0.1 37.1 AD- A- 1798 CACUGGAUAUGUUUG 2042- A- 1885 UGCAGUCAAACAU 2040- 57998 11108 ACUGCA 2062 1102662.1 AUCCAGUGAA 2062 1.1 38.1 AD- A- 1799 GGACCUUGUGUGAUC 2591- A- 1886 UGGUCUGAUCACA 2589- 57998 11108 AGACCA 2611 1103464.1 CAAGGUCCUC 2611 2.1 39.1 AD- A- 1800 UCAGACCAUUGAAAC 2604- A- 1887 UUAGUGGUUUCAA 2602- 57998 11108 CACUAA 2624 1103490.1 UGGUCUGAUC 2624 3.1 40.1 AD- A- 1801 CAUUGAAACCACUAA 2610- A- 1888 UCAGAAUUAGUGG 2608- 57998 11108 UUCUGA 2630 1103502.1 UUUCAAUGGU 2630 4.1 41.1 AD- A- 1802 CUAGAGCAGUUUUCC 3352- A- 1889 UAUCUCGGAAAAC 3350- 57998 11108 GAGAUA 3372 1103920.1 UGCUCUAGAG 3372 5.1 42.1 AD- A- 1803 UAGAGCAGUUUUCCG 3353- A- 1890 UUAUCUCGGAAAA 3351- 57998 11108 AGAUAA 3373 1103922.1 CUGCUCUAGA 3373 6.1 43.1 AD- A- 1804 AGUUUUCCGAGAUAU 3359- A- 1891 UACGGAAUAUCUC 3357- 57998 11108 UCCGUA 3379 1103934.1 GGAAAACUGC 3379 7.1 44.1 AD- A- 1805 GCGGAUCAAACCUCAC 1332- A- 1892 UUUUGGUGAGGUU 1330- 57998 11108 CAAAA 1352 1101748.1 UGAUCCGCAU 1352 8.1 45.1 AD- A- 1806 AGCAUUUGUUUGUCC 1463- A- 1893 UAUCUUGGACAAA 1461- 57998 11108 AAGAUA 1483 1101962.1 CAAAUGCUUU 1483 9.1 46.1 AD- A- 1807 UCUCACCGGAAAGACC 1635- A- 1894 UAAUCGGUCUUUC 1633- 57999 11108 GAUUA 1655 1102120.1 CGGUGAGAGG 1655 0.1 47.1 AD- A- 1808 UCUAGAGCAGUUUUC 3351- A- 1895 UUCUCGGAAAACU 3349- 57999 11108 CGAGAA 3371 1103918.1 GCUCUAGAGA 3371 2.1 49.1 AD- A- 1809 AUUGCACGGAAACUU  625- A- 1896 UACGAAAAGUUUC  623- 57999 11108 UUCGUA  645 1100955.1 CGUGCAAUCC  645 3.1 50.1 AD- A- 1810 GCUCUGGGAUUUGAU  127- A- 1897 UUGAAUAUCAAAU  125- 57999 11108 AUUCAA  147 1100507.1 CCCAGAGCAC  147 5.1 52.1 AD- A- 1811 UGAGCCUUGUUCAGA 1440- A- 1898 UUCCGCUCUGAAC 1438- 57999 11108 GCGGAA 1460 1101930.1 AAGGCUCACA 1460 6.1 53.1 AD- A- 1812 AGCCUUGUUCAGAGC 1442- A- 1899 UUCUCCGCUCUGA 1440- 57999 11108 GGAGAA 1462 1101934.1 ACAAGGCUCA 1462 7.1 54.1 AD- A- 1813 CAUUUGUUUGUCCAA 1465- A- 1900 UGGAUCUUGGACA 1463- 57999 11108 GAUCCA 1485 1101966.1 AACAAAUGCU 1485 8.1 55.1 AD- A- 1814 GGAAAGACCGAUUAA 1642- A- 1901 UCAUGGUUAAUCG 1640- 57999 11108 CCAUGA 1662 1102134.1 GUCUUUCCGG 1662 9.1 56.1 AD- A- 1815 GAAAGACCGAUUAAC 1643- A- 1902 UACAUGGUUAAUC 1641- 58000 11108 CAUGUA 1663 1102136.1 GGUCUUUCCG 1663 0.1 57.1 AD- A- 1816 AAGACCGAUUAACCA 1645- A- 1903 UUGACAUGGUUAA 1643- 58000 11108 UGUCAA 1665 1102140.1 UCGGUCUUUC 1665 1.1 58.1 AD- A- 1817 GACCGAUUAACCAUG 1647- A- 1904 UGGUGACAUGGUU 1645- 58000 11108 UCACCA 1667 1102144.1 AAUCGGUCUU 1667 2.1 59.1

TABLE 8A Exemplary Human VEGF-A siRNA Modified Single Strands and Duplex Sequences Anti- SEQ ID SEQ ID SEQ sense NO: NO: Duplex Sense Oligo ID NO: Oligo (Anti- mRNA Target (mRNA Name Name (Sense) Sense Sequence Name sense) Antisense Sequence Sequence target) AD- A- 2000 csgsgugcUfgGfAfAfuuuga A- 2449 VPusAfsauaUfcaaauucC CUCGGUGCUGGAAU 4700 12228 22821 uauuaL96 228211 faGfcaccgsasg UUGAUAUUC 66.1 11.1 2.1 AD- A- 2001 gsgsugcuGfgAfAfUfuugau A- 2450 VPusGfsaauAfucaaauuC UCGGUGCUGGAAUU 4701 12228 22821 auucaL96 228211 fcAfgcaccsgsa UGAUAUUCA 67.1 13.1 4.1 AD- A- 2002 usgscuggAfaUfUfUfgauauu A- 2451 VPusAfsugaAfuaucaaaU GGUGCUGGAAUUUG 4702 12228 22821 cauaL96 228211 fuCfcagcascsc AUAUUCAUU 68.1 15.1 6.1 AD- A- 2003 gscsuggaAfuUfUfGfauauuc A- 2452 VPusAfsaugAfauaucaaA GUGCUGGAAUUUGA 4703 12228 22821 auuaL96 228211 fuUfccagcsasc UAUUCAUUG 69.1 17.1 8.1 AD- A- 2004 usgsgaauUfuGfAfUfauucau A- 2453 VPusUfscaaUfgaauaucA GCUGGAAUUUGAUA 4704 12228 22821 ugaaL96 228212 faAfuuccasgsc UUCAUUGAU 70.1 19.1 0.1 AD- A- 2005 gsgsaauuUfgAfUfAfuucauu A- 2454 VPusAfsucaAfugaauauC CUGGAAUUUGAUAU 4705 12228 22821 gauaL96 228212 faAfauuccsasg UCAUUGAUC 71.1 21.1 2.1 AD- A- 2006 gsasauuuGfaUfAfUfucauug A- 2455 VPusGfsaucAfaugaauaU UGGAAUUUGAUAU 4706 12228 22821 aucaL96 228212 fcAfaauucscsa UCAUUGAUCC 72.1 23.1 4.1 AD- A- 2007 asasuuugAfuAfUfUfcauuga A- 2456 VPusGfsgauCfaaugaauA GGAAUUUGAUAUUC 4707 12228 22821 uccaL96 228212 fuCfaaauuscsc AUUGAUCCG 73.1 25.1 6.1 AD- A- 2008 asusuugaUfaUfUfCfauugau A- 2457 VPusCfsggaUfcaaugaaU GAAUUUGAUAUUCA 4708 12228 22821 ccgaL96 228212 faUfcaaaususc UUGAUCCGG 74.1 27.1 8.1 AD- A- 2009 ususugauAfuUfCfAfuugauc A- 2458 VPusCfscggAfucaaugaA AAUUUGAUAUUCAU 4709 12228 22821 cggaL96 228213 fuAfucaaasusu UGAUCCGGG 75.1 29.1 0.1 AD- A- 2010 ususuauuUfuUfGfCfuugcca A- 2459 VPusGfsaauGfgcaagcaA AAUUUAUUUUUGCU 4710 12228 22821 uucaL96 228213 faAfauaaasusu UGCCAUUCC 76.1 31.1 2.1 AD- A- 2011 ususauuuUfuGfCfUfugccau A- 2460 VPusGfsgaaUfggcaagcA AUUUAUUUUUGCUU 4711 12228 22821 uccaL96 228213 faAfaauaasasu GCCAUUCCC 77.1 33.1 4.1 AD- A- 2012 csasaaucAfcUfGfUfggauuu A- 2461 VPusCfscaaAfauccacaG CCCAAAUCACUGUG 4712 12228 22821 uggaL96 228213 fuGfauuugsgsg GAUUUUGGA 78.1 35.1 6.1 AD- A- 2013 asasaucaCfuGfUfGfgauuuu A- 2462 VPusUfsccaAfaauccacA CCAAAUCACUGUGG 4713 12228 22821 ggaaL96 228213 fgUfgauuusgsg AUUUUGGAA 79.1 37.1 8.1 AD- A- 2014 asasucacUfgUfGfGfauuuug A- 2463 VPusUfsuccAfaaauccaC CAAAUCACUGUGGA 4714 12228 22821 gaaaL96 228214 faGfugauususg UUUUGGAAA 80.1 39.1 0.1 AD- A- 2015 asuscacuGfuGfGfAfuuuugg A- 2464 VPusUfsuucCfaaaauccA AAAUCACUGUGGAU 4715 12228 22821 aaaaL96 228214 fcAfgugaususu UUUGGAAAC 81.1 41.1 2.1 AD- A- 2016 uscsacugUfgGfAfUfuuugga A- 2465 VPusGfsuuuCfcaaaaucC AAUCACUGUGGAUU 4716 12228 22821 aacaL96 228214 faCfagugasusu UUGGAAACC 82.1 43.1 4.1 AD- A- 2017 csascuguGfgAfUfUfuuggaa A- 2466 VPusGfsguuUfccaaaauC AUCACUGUGGAUUU 4717 12228 22821 accaL96 228214 fcAfcagugsasu UGGAAACCA 83.1 45.1 6.1 AD- A- 2018 ascsugugGfaUfUfUfuggaaa A- 2467 VPusUfsgguUfuccaaaaU UCACUGUGGAUUUU 4718 12228 22821 ccaaL96 228214 fcCfacagusgsa GGAAACCAG 84.1 47.1 8.1 AD- A- 2019 csusguggAfuUfUfUfggaaac A- 2468 VPusCfsuggUfuuccaaaA CACUGUGGAUUUUG 4719 12228 22821 cagaL96 228215 fuCfcacagsusg GAAACCAGC 85.1 49.1 0.1 AD- A- 2020 usgsuggaUfuUfUfGfgaaacc A- 2469 VPusGfscugGfuuuccaa ACUGUGGAUUUUGG 4720 12228 22821 agcaL96 228215 AfaUfccacasgsu AAACCAGCA 86.1 51.1 2.1 AD- A- 2021 gsusggauUfuUfGfGfaaacca A- 2470 VPusUfsgcuGfguuucca CUGUGGAUUUUGGA 4721 12228 22821 gcaaL96 228215 AfaAfuccacsasg AACCAGCAG 87.1 53.1 4.1 AD- A- 2022 gsasuuuuGfgAfAfAfccagca A- 2471 VPusUfsucuGfcugguuu UGGAUUUUGGAAAC 4722 12228 22821 gaaaL96 228215 CfcAfaaaucscsa CAGCAGAAA 88.1 55.1 6.1 AD- A- 2023 asusuuugGfaAfAfCfcagcag A- 2472 VPusUfsuucUfgcugguu GGAUUUUGGAAACC 4723 12228 22821 aaaaL96 228215 UfcCfaaaauscsc AGCAGAAAG 89.1 57.1 8.1 AD- A- 2024 ususuuggAfaAfCfCfagcaga A- 2473 VPusCfsuuuCfugcuggu GAUUUUGGAAACCA 4724 12228 22821 aagaL96 228216 UfuCfcaaaasusc GCAGAAAGA 90.1 59.1 0.1 AD- A- 2025 ususuggaAfaCfCfAfgcagaa A- 2474 VPusUfscuuUfcugcugg AUUUUGGAAACCAG 4725 12228 22821 agaaL96 228216 UfuUfccaaasasu CAGAAAGAG 91.1 61.1 2.1 AD- A- 2026 gsgsaaacCfaGfCfAfgaaaga A- 2475 VPusUfsccuCfuuucugc UUGGAAACCAGCAG 4726 12228 22821 ggaaL96 228216 UfgGfuuuccsasa AAAGAGGAA 92.1 63.1 4.1 AD- A- 2027 asasaccaGfcAfGfAfaagagg A- 2476 VPusUfsuucCfucuuucu GGAAACCAGCAGAA 4727 12228 22821 aaaaL96 228216 GfcUfgguuuscsc AGAGGAAAG 93.1 65.1 6.1 AD- A- 2028 asasccagCfaGfAfAfagagga A- 2477 VPusCfsuuuCfcucuuuc GAAACCAGCAGAAA 4728 12228 22821 aagaL96 228216 UfgCfugguususc GAGGAAAGA 94.1 67.1 8.1 AD- A- 2029 ascscagcAfgAfAfAfgaggaa A- 2478 VPusUfscuuUfccucuuu AAACCAGCAGAAAG 4729 12228 22821 agaaL96 228217 CfuGfcuggususu AGGAAAGAG 95.1 69.1 0.1 AD- A- 2030 cscsagcaGfaAfAfGfaggaaa A- 2479 VPusCfsucuUfuccucuu AACCAGCAGAAAGA 4730 12228 22821 gagaL96 228217 UfcUfgcuggsusu GGAAAGAGG 96.1 71.1 2.1 AD- A- 2031 csasgcagAfaAfGfAfggaaag A- 2480 VPusCfscucUfuuccucuU ACCAGCAGAAAGAG 4731 12228 22821 aggaL96 228217 fuCfugcugsgsu GAAAGAGGU 97.1 73.1 4.1 AD- A- 2032 asgscagaAfaGfAfGfgaaaga A- 2481 VPusAfsccuCfuuuccucU CCAGCAGAAAGAGG 4732 12228 22821 gguaL96 228217 fuUfcugcusgsg AAAGAGGUA 98.1 75.1 6.1 AD- A- 2033 gscsagaaAfgAfGfGfaaagag A- 2482 VPusUfsaccUfcuuuccuC CAGCAGAAAGAGGA 4733 12228 22821 guaaL96 228217 fuUfucugcsusg AAGAGGUAG 99.1 77.1 8.1 AD- A- 2034 csasgaaaGfaGfGfAfaagagg A- 2483 VPusCfsuacCfucuuuccU AGCAGAAAGAGGAA 4734 12229 22821 uagaL96 228218 fcUfuucugscsu AGAGGUAGC 00.1 79.1 0.1 AD- A- 2035 asasagagGfaAfAfGfagguag A- 2484 VPusUfsugcUfaccucuu AGAAAGAGGAAAG 4735 12229 22821 caaaL96 228218 UfcCfucuuuscsu AGGUAGCAAG 01.1 81.1 2.1 AD- A- 2036 asasgaggAfaAfGfAfgguagc A- 2485 VPusCfsuugCfuaccucuU GAAAGAGGAAAGA 4736 12229 22821 aagaL96 228218 fuCfcucuususc GGUAGCAAGA 02.1 83.1 4.1 AD- A- 2037 asgsaggaAfaGfAfGfguagca A- 2486 VPusUfscuuGfcuaccucU AAAGAGGAAAGAG 4737 12229 22821 agaaL96 228218 fuUfccucususu GUAGCAAGAG 03.1 85.1 6.1 AD- A- 2038 gsasggaaAfgAfGfGfuagcaa A- 2487 VPusCfsucuUfgcuaccuC AAGAGGAAAGAGG 4738 12229 22821 gagaL96 228218 fuUfuccucsusu UAGCAAGAGC 04.1 87.1 8.1 AD- A- 2039 gsgsaaagAfgGfUfAfgcaaga A- 2488 VPusAfsgcuCfuugcuacC GAGGAAAGAGGUA 4739 12229 22821 gcuaL96 228219 fuCfuuuccsusc GCAAGAGCUC 05.1 89.1 0.1 AD- A- 2040 asgsguagCfaAfGfAfgcucca A- 2489 VPusCfsucuGfgagcucu AGAGGUAGCAAGAG 4740 12229 22821 gagaL96 228219 UfgCfuaccuscsu CUCCAGAGA 06.1 91.1 2.1 AD- A- 2041 uscscagaGfaGfAfAfgucgag A- 2490 VPusUfsuccUfcgacuucU GCUCCAGAGAGAAG 4741 12229 22821 gaaaL96 228219 fcUfcuggasgsc UCGAGGAAG 07.1 93.1 4.1 AD- A- 2042 cscsagagAfgAfAfGfucgagg A- 2491 VPusCfsuucCfucgacuuC CUCCAGAGAGAAGU 4742 12229 22821 aagaL96 228219 fuCfucuggsasg CGAGGAAGA 08.1 95.1 6.1 AD- A- 2043 csasgagaGfaAfGfUfcgagga A- 2492 VPusUfscuuCfcucgacuU UCCAGAGAGAAGUC 4743 12229 22821 agaaL96 228219 fcUfcucugsgsa GAGGAAGAG 09.1 97.1 8.1 AD- A- 2044 asgsagaaGfuCfGfAfggaaga A- 2493 VPusCfsucuCfuuccucgA AGAGAGAAGUCGAG 4744 12229 22821 gagaL96 228220 fcUfucucuscsu GAAGAGAGA 10.1 99.1 0.1 AD- A- 2045 gsasgaagUfcGfAfGfgaagag A- 2494 VPusUfscucUfcuuccucG GAGAGAAGUCGAGG 4745 12229 22822 agaaL96 228220 faCfuucucsusc AAGAGAGAG 11.1 01.1 2.1 AD- A- 2046 gsasagucGfaGfGfAfagagag A- 2495 VPusUfscucUfcucuuccU GAGAAGUCGAGGAA 4746 12229 22822 agaaL96 228220 fcGfacuucsusc GAGAGAGAC 12.1 03.1 4.1 AD- A- 2047 asgsugagUfgAfCfCfugcuuu A- 2496 VPusCfscaaAfagcagguC AAAGUGAGUGACCU 4747 12229 22822 uggaL96 228220 faCfucacususu GCUUUUGGG 13.1 05.1 6.1 AD- A- 2048 gsgscgucGfcAfCfUfgaaacu A- 2497 VPusAfsaaaGfuuucagu GCGGCGUCGCACUG 4748 12229 22822 uuuaL96 228220 GfcGfacgccsgsc AAACUUUUC 14.1 07.1 8.1 AD- A- 2049 gsuscgcaCfuGfAfAfacuuuu A- 2498 VPusAfscgaAfaaguuuc GCGUCGCACUGAAA 4749 12229 22822 cguaL96 228221 AfgUfgcgacsgsc CUUUUCGUC 15.1 09.1 0.1 AD- A- 2050 uscsgcacUfgAfAfAfcuuuuc A- 2499 VPusGfsacgAfaaaguuuC CGUCGCACUGAAAC 4750 12229 22822 gucaL96 228221 faGfugcgascsg UUUUCGUCC 16.1 11.1 2.1 AD- A- 2051 csascugaAfaCfUfUfuucguc A- 2500 VPusUfsuggAfcgaaaag CGCACUGAAACUUU 4751 12229 22822 caaaL96 228221 UfuUfcagugscsg UCGUCCAAC 17.1 13.1 4.1 AD- A- 2052 ascsugaaAfcUfUfUfucgucc A- 2501 VPusGfsuugGfacgaaaaG GCACUGAAACUUUU 4752 12229 22822 aacaL96 228221 fuUfucagusgsc CGUCCAACU 18.1 15.1 6.1 AD- A- 2053 usgsaaacUfuUfUfCfguccaa A- 2502 VPusAfsaguUfggacgaa ACUGAAACUUUUCG 4753 12229 22822 cuuaL96 228222 AfaGfuuucasgsu UCCAACUUC 20.1 19.1 0.1 AD- A- 2054 asascuuuUfcGfUfCfcaacuu A- 2503 VPusCfsagaAfguuggac GAAACUUUUCGUCC 4754 12229 22822 cugaL96 228222 GfaAfaaguususc AACUUCUGG 21.1 21.1 2.1 AD- A- 2055 csusgggcUfgUfUfCfucgcuu A- 2504 VPusCfscgaAfgcgagaaC UUCUGGGCUGUUCU 4755 12229 22822 cggaL96 228222 faGfcccagsasa CGCUUCGGA 22.1 23.1 4.1 AD- A- 2056 usgsggcuGfuUfCfUfcgcuuc A- 2505 VPusUfsccgAfagcgagaA UCUGGGCUGUUCUC 4756 12229 22822 ggaaL96 228222 fcAfgcccasgsa GCUUCGGAG 23.1 25.1 6.1 AD- A- 2057 gsgsgcugUfuCfUfCfgcuucg A- 2506 VPusCfsuccGfaagcgagA CUGGGCUGUUCUCG 4757 12229 22822 gagaL96 228222 faCfagcccsasg CUUCGGAGG 24.1 27.1 8.1 AD- A- 2058 gscsuguuCfuCfGfCfuucgga A- 2507 VPusUfsccuCfcgaagcgA GGGCUGUUCUCGCU 4758 12229 22822 ggaaL96 228223 fgAfacagcscsc UCGGAGGAG 25.1 29.1 0.1 AD- A- 2059 gscscgcgAfgAfAfGfugcuag A- 2508 VPusGfsagcUfagcacuuC GAGCCGCGAGAAGU 4759 12229 22822 cucaL96 228223 fuCfgcggcsusc GCUAGCUCG 26.1 31.1 2.1 AD- A- 2060 cscsgcgaGfaAfGfUfgcuagc A- 2509 VPusCfsgagCfuagcacuU AGCCGCGAGAAGUG 4760 12229 22822 ucgaL96 228223 fcUfcgcggscsu CUAGCUCGG 27.1 33.1 4.1 AD- A- 2061 gscscuccGfaAfAfCfcaugaa A- 2510 VPusAfsaguUfcaugguu GGGCCUCCGAAACC 4761 12229 22822 cuuaL96 228223 UfcGfgaggcscsc AUGAACUUU 28.1 35.1 6.1 AD- A- 2062 asasggagGfaGfGfGfcagaau A- 2511 VPusAfsugaUfucugccc AGAAGGAGGAGGGC 4762 12229 22822 cauaL96 228223 UfcCfuccuuscsu AGAAUCAUC 29.1 37.1 8.1 AD- A- 2063 gsgscagaAfuCfAfUfcacgaa A- 2512 VPusCfsacuUfcgugaug AGGGCAGAAUCAUC 4763 12229 22822 gugaL96 228224 AfuUfcugccscsu ACGAAGUGG 30.1 39.1 0.1 AD- A- 2064 asasucauCfaCfGfAfaguggu A- 2513 VPusUfsucaCfcacuucgU AGAAUCAUCACGAA 4764 12229 22822 gaaaL96 228224 fgAfugauuscsu GUGGUGAAG 31.1 41.1 2.1 AD- A- 2065 asuscaucAfcGfAfAfguggug A- 2514 VPusCfsuucAfccacuucG GAAUCAUCACGAAG 4765 12229 22822 aagaL96 228224 fuGfaugaususc UGGUGAAGU 32.1 43.1 4.1 AD- A- 2066 uscsaucaCfgAfAfGfugguga A- 2515 VPusAfscuuCfaccacuuC AAUCAUCACGAAGU 4766 12229 22822 aguaL96 228224 fgUfgaugasusu GGUGAAGUU 33.1 45.1 6.1 AD- A- 2067 csasucacGfaAfGfUfggugaa A- 2516 VPusAfsacuUfcaccacuU AUCAUCACGAAGUG 4767 12229 22822 guuaL96 228224 fcGfugaugsasu GUGAAGUUC 34.1 47.1 8.1 AD- A- 2068 uscsacgaAfgUfGfGfugaagu A- 2517 VPusUfsgaaCfuucaccaC CAUCACGAAGUGGU 4768 12229 22822 ucaaL96 228225 fuUfcgugasusg GAAGUUCAU 35.1 49.1 0.1 AD- A- 2069 asasguggUfgAfAfGfuucaug A- 2518 VPusAfsuccAfugaacuuC CGAAGUGGUGAAGU 4769 12229 22822 gauaL96 228225 faCfcacuuscsg UCAUGGAUG 36.1 51.1 2.1 AD- A- 2070 asgsugguGfaAfGfUfucaugg A- 2519 VPusCfsaucCfaugaacuU GAAGUGGUGAAGU 4770 12229 22822 augaL96 228225 fcAfccacususc UCAUGGAUGU 37.1 53.1 4.1 AD- A- 2071 gsusggugAfaGfUfUfcaugga A- 2520 VPusAfscauCfcaugaacU AAGUGGUGAAGUUC 4771 12229 22822 uguaL96 228225 fuCfaccacsusu AUGGAUGUC 38.1 55.1 6.1 AD- A- 2072 gsgsugaaGfuUfCfAfuggaug A- 2521 VPusAfsgacAfuccaugaA GUGGUGAAGUUCAU 4772 12229 22822 ucuaL96 228225 fcUfucaccsasc GGAUGUCUA 39.1 57.1 8.1 AD- A- 2073 gsusgaagUfuCfAfUfggaugu A- 2522 VPusUfsagaCfauccaugA UGGUGAAGUUCAUG 4773 12229 22822 cuaaL96 228226 faCfuucacscsa GAUGUCUAU 40.1 59.1 0.1 AD- A- 2074 usgsaaguUfcAfUfGfgauguc A- 2523 VPusAfsuagAfcauccauG GGUGAAGUUCAUGG 4774 12229 22822 uauaL96 228226 faAfcuucascsc AUGUCUAUC 41.1 61.1 2.1 AD- A- 2075 asasguucAfuGfGfAfugucua A- 2524 VPusUfsgauAfgacauccA UGAAGUUCAUGGAU 4775 12229 22822 ucaaL96 228226 fuGfaacuuscsa GUCUAUCAG 42.1 63.1 4.1 AD- A- 2076 asgsuucaUfgGfAfUfgucuau A- 2525 VPusCfsugaUfagacaucC GAAGUUCAUGGAUG 4776 12229 22822 cagaL96 228226 faUfgaacususc UCUAUCAGC 43.1 65.1 6.1 AD- A- 2077 ususcaugGfaUfGfUfcuauca A- 2526 VPusCfsgcuGfauagacaU AGUUCAUGGAUGUC 4777 12229 22822 gcgaL96 228226 fcCfaugaascsu UAUCAGCGC 44.1 67.1 8.1 AD- A- 2078 gsusggacAfuCfUfUfccagga A- 2527 VPusUfsacuCfcuggaagA UGGUGGACAUCUUC 4778 12229 22822 guaaL96 228227 fuGfuccacscsa CAGGAGUAC 45.1 69.1 0.1 AD- A- 2079 uscsgaguAfcAfUfCfuucaag A- 2528 VPusUfsggcUfugaagau GAUCGAGUACAUCU 4779 12229 22822 ccaaL96 228227 GfuAfcucgasusc UCAAGCCAU 46.1 71.1 2.1 AD- A- 2080 csgsaguaCfaUfCfUfucaagc A- 2529 VPusAfsuggCfuugaaga AUCGAGUACAUCUU 4780 12229 22822 cauaL96 228227 UfgUfacucgsasu CAAGCCAUC 47.1 73.1 4.1 AD- A- 2081 gsasguacAfuCfUfUfcaagcc A- 2530 VPusGfsaugGfcuugaag UCGAGUACAUCUUC 4781 12229 22822 aucaL96 228227 AfuGfuacucsgsa AAGCCAUCC 48.1 75.1 6.1 AD- A- 2082 gsusacauCfuUfCfAfagccau A- 2531 VPusAfsggaUfggcuuga GAGUACAUCUUCAA 4782 12229 22822 ccuaL96 228227 AfgAfuguacsusc GCCAUCCUG 49.1 77.1 8.1 AD- A- 2083 cscsaacaUfcAfCfCfaugcaga A- 2532 VPusAfsaucUfgcauggu GUCCAACAUCACCA 4783 12229 22822 uuaL96 228228 GfaUfguuggsasc UGCAGAUUA 50.1 79.1 0.1 AD- A- 2084 csasacauCfaCfCfAfugcaga A- 2533 VPusUfsaauCfugcaugg UCCAACAUCACCAU 4784 12229 22822 uuaaL96 228228 UfgAfuguugsgsa GCAGAUUAU 51.1 81.1 2.1 AD- A- 2085 ascsaucaCfAfUfGfcagauu A- 2534 VPusCfsauaAfucugcauG CAACAUCACCAUGC 4785 12229 22822 augaL96 228228 fgUfgaugususg AGAUUAUGC 52.1 83.1 4.1 AD- A- 2086 csasucacCfaUfGfCfagauua A- 2535 VPusGfscauAfaucugcaU AACAUCACCAUGCA 4786 12229 22822 ugcaL96 228228 fgGfugaugsusu GAUUAUGCG 53.1 85.1 6.1 AD- A- 2087 ascscaugCfaGfAfUfuaugcg A- 2536 VPusAfsuccGfcauaaucU UCACCAUGCAGAUU 4787 12229 22822 gauaL96 228228 fgCfauggusgsa AUGCGGAUC 54.1 87.1 8.1 AD- A- 2088 asusgcagAfuUfAfUfgcggau A- 2537 VPusUfsugaUfccgcauaA CCAUGCAGAUUAUG 4788 12229 22822 caaaL96 228229 fuCfugcausgsg CGGAUCAAA 55.1 89.1 0.1 AD- A- 2089 usgscagaUfuAfUfGfcggauc A- 2538 VPusUfsuugAfuccgcau CAUGCAGAUUAUGC 4789 12229 22822 aaaaL96 228229 AfaUfcugcasusg GGAUCAAAC 56.1 91.1 2.1 AD- A- 2090 gscsagauUfaUfGfCfggauca A- 2539 VPusGfsuuuGfauccgca AUGCAGAUUAUGCG 4790 12229 22822 aacaL96 228229 UfaAfucugcsasu GAUCAAACC 57.1 93.1 4.1 AD- A- 2091 gsasuuauGfcGfGfAfucaaac A- 2540 VPusGfsaggUfuugaucc CAGAUUAUGCGGAU 4791 12229 22822 cucaL96 228229 GfcAfuaaucsusg CAAACCUCA 58.1 95.1 6.1 AD- A- 2092 asusuaugCfgGfAfUfcaaacc A- 2541 VPusUfsgagGfuuugauc AGAUUAUGCGGAUC 4792 12229 22822 ucaaL96 228229 CfgCfauaauscsu AAACCUCAC 59.1 97.1 8.1 AD- A- 2093 csgsgaucAfaAfCfCfucacca A- 2542 VPusCfscuuGfgugaggu UGCGGAUCAAACCU 4793 12229 22822 aggaL96 228230 UfuGfauccgscsa CACCAAGGC 60.1 99.1 0.1 AD- A- 2094 gsgsagagAfuGfAfGfcuuccu A- 2543 VPusUfsguaGfgaagcuc UAGGAGAGAUGAGC 4794 12229 22823 acaaL96 228230 AfuCfucuccsusa UUCCUACAG 61.1 01.1 2.1 AD- A- 2095 gsasgaugAfgCfUfUfccuaca A- 2544 VPusUfsgcuGfuaggaag GAGAGAUGAGCUUC 4795 12229 22823 gcaaL96 228230 CfuCfaucucsusc CUACAGCAC 62.1 03.1 4.1 AD- A- 2096 gsasugagCfuUfCfCfuacagc A- 2545 VPusUfsgugCfuguagga GAGAUGAGCUUCCU 4796 12229 22823 acaaL96 228230 AfgCfucaucsusc ACAGCACAA 63.1 05.1 6.1 AD- A- 2097 asusgagcUfuCfCfUfacagca A- 2546 VPusUfsuguGfcuguagg AGAUGAGCUUCCUA 4797 12229 22823 caaaL96 228230 AfaGfcucauscsu CAGCACAAC 64.1 07.1 8.1 AD- A- 2098 gsasgcuuCfcUfAfCfagcaca A- 2547 VPusUfsguuGfugcugua AUGAGCUUCCUACA 4798 12229 22823 acaaL96 228231 GfgAfagcucsasu GCACAACAA 65.1 09.1 0.1 AD- A- 2099 asgscuucCfuAfCfAfgcacaa A- 2548 VPusUfsuguUfgugcugu UGAGCUUCCUACAG 4799 12229 22823 caaaL96 228231 AfgGfaagcuscsa CACAACAAA 66.1 11.1 2.1 AD- A- 2100 gscsuuccUfaCfAfGfcacaac A- 2549 VPusUfsuugUfugugcug GAGCUUCCUACAGC 4800 12229 22823 aaaaL96 228231 UfaGfgaagcsusc ACAACAAAU 67.1 13.1 4.1 AD- A- 2101 csusuccuAfcAfGfCfacaaca A- 2550 VPusAfsuuuGfuugugcu AGCUUCCUACAGCA 4801 12229 22823 aauaL96 228231 GfuAfggaagscsu CAACAAAUG 68.1 15.1 6.1 AD- A- 2102 uscscuacAfgCfAfCfaacaaa A- 2551 VPusAfscauUfuguugug CUUCCUACAGCACA 4802 12229 22823 uguaL96 228231 CfuGfuaggasasg ACAAAUGUG 69.1 17.1 8.1 AD- A- 2103 csusacagCfaCfAfAfcaaaug A- 2552 VPusUfscacAfuuuguug UCCUACAGCACAAC 4803 12229 22823 ugaaL96 228232 UfgCfuguagsgsa AAAUGUGAA 70.1 19.1 0.1 AD- A- 2104 usascagcAfcAfAfCfaaaugu A- 2553 VPusUfsucaCfauuuguu CCUACAGCACAACA 4804 12229 22823 gaaaL96 228232 GfuGfcuguasgsg AAUGUGAAU 71.1 21.1 2.1 AD- A- 2105 asgscacaAfcAfAfAfugugaa A- 2554 VPusGfscauUfcacauuuG ACAGCACAACAAAU 4805 12229 22823 ugcaL96 228232 fuUfgugcusgsu GUGAAUGCA 72.1 23.1 4.1 AD- A- 2106 gscsacaaCfaAfAfUfgugaau A- 2555 VPusUfsgcaUfucacauuU CAGCACAACAAAUG 4806 12229 22823 gcaaL96 228232 fgUfugugcsusg UGAAUGCAG 73.1 25.1 6.1 AD- A- 2107 csuscaccAfgGfAfAfagacug A- 2556 VPusUfsaucAfgucuuuc CUCUCACCAGGAAA 4807 12229 22823 auaaL96 228232 CfuGfgugagsasg GACUGAUAC 74.1 27.1 8.1 AD- A- 2108 uscsaccaGfgAfAfAfgacuga A- 2557 VPusGfsuauCfagucuuu UCUCACCAGGAAAG 4808 12229 22823 uacaL96 228233 CfcUfggugasgsa ACUGAUACA 75.1 29.1 0.1 AD- A- 2109 csasccagGfaAfAfGfacugau A- 2558 VPusUfsguaUfcagucuu CUCACCAGGAAAGA 4809 12229 22823 acaaL96 228233 UfcCfuggugsasg CUGAUACAG 76.1 31.1 2.1 AD- A- 2110 ascscaggAfaAfGfAfcugaua A- 2559 VPusCfsuguAfucagucu UCACCAGGAAAGAC 4810 12229 22823 cagaL96 228233 UfuCfcuggusgsa UGAUACAGA 77.1 33.1 4.1 AD- A- 2111 cscsaggaAfaGfAfCfugauac A- 2560 VPusUfscugUfaucaguc CACCAGGAAAGACU 4811 12229 22823 agaaL96 228233 UfuUfccuggsusg GAUACAGAA 78.1 35.1 6.1 AD- A- 2112 csasggaaAfgAfCfUfgauaca A- 2561 VPusUfsucuGfuaucagu ACCAGGAAAGACUG 4812 12229 22823 gaaaL96 228233 CfuUfuccugsgsu AUACAGAAC 79.1 37.1 8.1 AD- A- 2113 asgsgaaaGfaCfUfGfauacag A- 2562 VPusGfsuucUfguaucag CCAGGAAAGACUGA 4813 12229 22823 aacaL96 228234 UfcUfuuccusgsg UACAGAACG 80.1 39.1 0.1 AD- A- 2114 gsgsaaagAfcUfGfAfuacaga A- 2563 VPusCfsguuCfuguauca CAGGAAAGACUGAU 4814 12229 22823 acgaL96 228234 GfuCfuuuccsusg ACAGAACGA 81.1 41.1 2.1 AD- A- 2115 gsasaagaCfuGfAfUfacagaa A- 2564 VPusUfscguUfcuguauc AGGAAAGACUGAUA 4815 12229 22823 cgaaL96 228234 AfgUfcuuucscsu CAGAACGAU 82.1 43.1 4.1 AD- A- 2116 asasagacUfgAfUfAfcagaac A- 2565 VPusAfsucgUfucuguau GGAAAGACUGAUAC 4816 12229 22823 gauaL96 228234 CfaGfucuuuscsc AGAACGAUC 83.1 45.1 6.1 AD- A- 2117 asasgacuGfaUfAfCfagaacg A- 2566 VPusGfsaucGfuucugua GAAAGACUGAUACA 4817 12229 22823 aucaL96 228234 UfcAfgucuususc GAACGAUCG 84.1 47.1 8.1 AD- A- 2118 asgsacugAfuAfCfAfgaacga A- 2567 VPusCfsgauCfguucugu AAAGACUGAUACAG 4818 12229 22823 ucgaL96 228235 AfuCfagucususu AACGAUCGA 85.1 49.1 0.1 AD- A- 2119 gsascugaUfaCfAfGfaacgau A- 2568 VPusUfscgaUfcguucug AAGACUGAUACAGA 4819 12229 22823 cgaaL96 228235 UfaUfcagucsusu ACGAUCGAU 86.1 51.1 2.1 AD- A- 2120 ascsugauAfcAfGfAfacgauc A- 2569 VPusAfsucgAfucguucu AGACUGAUACAGAA 4820 12229 22823 gauaL96 228235 GfuAfucaguscsu CGAUCGAUA 87.1 53.1 4.1 AD- A- 2121 csusgauaCfaGfAfAfcgaucg A- 2570 VPusUfsaucGfaucguuc GACUGAUACAGAAC 4821 12229 22823 auaaL96 228235 UfgUfaucagsusc GAUCGAUAC 88.1 55.1 6.1 AD- A- 2122 usgsauacAfgAfAfCfgaucga A- 2571 VPusGfsuauCfgaucguu ACUGAUACAGAACG 4822 12229 22823 uacaL96 228235 CfuGfuaucasgsu AUCGAUACA 89.1 57.1 8.1 AD- A- 2123 gsasuacaGfaAfCfGfaucgau A- 2572 VPusUfsguaUfcgaucgu CUGAUACAGAACGA 4823 12229 22823 acaaL96 228236 UfcUfguaucsasg UCGAUACAG 90.1 59.1 0.1 AD- A- 2124 asusacagAfaCfGfAfucgaua A- 2573 VPusCfsuguAfucgaucg UGAUACAGAACGAU 4824 12229 22823 cagaL96 228236 UfuCfuguauscsa CGAUACAGA 91.1 61.1 2.1 AD- A- 2125 usascagaAfcGfAfUfcgauac A- 2574 VPusUfscugUfaucgauc GAUACAGAACGAUC 4825 12229 22823 agaaL96 228236 GfuUfcuguasusc GAUACAGAA 92.1 63.1 4.1 AD- A- 2126 ascsagaaCfgAfUfCfgauaca A- 2575 VPusUfsucuGfuaucgau AUACAGAACGAUCG 4826 12229 22823 gaaaL96 228236 CfgUfucugusasu AUACAGAAA 93.1 65.1 6.1 AD- A- 2127 csasgaacGfaUfCfGfauacag A- 2576 VPusUfsuucUfguaucga UACAGAACGAUCGA 4827 12229 22823 aaaaL96 228236 UfcGfuucugsusa UACAGAAAC 94.1 67.1 8.1 AD- A- 2128 asgsaacgAfuCfGfAfuacaga A- 2577 VPusGfsuuuCfuguaucg ACAGAACGAUCGAU 4828 12229 22823 aacaL96 228237 AfuCfguucusgsu ACAGAAACC 95.1 69.1 0.1 AD- A- 2129 gsasacgaUfcGfAfUfacagaa A- 2578 VPusGfsguuUfcuguauc CAGAACGAUCGAUA 4829 12229 22823 accaL96 228237 GfaUfcguucsusg CAGAAACCA 96.1 71.1 2.1 AD- A- 2130 asascgauCfgAfUfAfcagaaa A- 2579 VPusUfsgguUfucuguau AGAACGAUCGAUAC 4830 12229 22823 ccaaL96 228237 CfgAfucguuscsu AGAAACCAC 97.1 73.1 4.1 AD- A- 2131 ascsgaucGfaUfAfCfagaaac A- 2580 VPusGfsuggUfuucugua GAACGAUCGAUACA 4831 12229 22823 cacaL96 228237 UfcGfaucgususc GAAACCACG 98.1 75.1 6.1 AD- A- 2132 csgsaucgAfuAfCfAfgaaacc A- 2581 VPusCfsgugGfuuucugu AACGAUCGAUACAG 4832 12229 22823 acgaL96 228237 AfuCfgaucgsusu AAACCACGC 99.1 77.1 8.1 AD- A- 2133 csasccauCfaCfCfAfucgacag A- 2582 VPusUfsucuGfucgaugg CACACCAUCACCAU 4833 12230 22823 aaaL96 228238 UfgAfuggugsusg CGACAGAAC 00.1 79.1 0.1 AD- A- 2134 csasucacCfaUfCfGfacagaac A- 2583 VPusCfsuguUfcugucga ACCAUCACCAUCGA 4834 12230 22823 agaL96 228238 UfgGfugaugsgsu CAGAACAGU 01.1 81.1 2.1 AD- A- 2135 uscsaccaUfcGfAfCfagaaca A- 2584 VPusGfsacuGfuucuguc CAUCACCAUCGACA 4835 12230 22823 gucaL96 228238 GfaUfggugasusg GAACAGUCC 02.1 83.1 4.1 AD- A- 2136 csasccauCfgAfCfAfgaacag A- 2585 VPusGfsgacUfguucugu AUCACCAUCGACAG 4836 12230 22823 uccaL96 228238 CfgAfuggugsasu AACAGUCCU 03.1 85.1 6.1 AD- A- 2137 ascscaucGfaCfAfGfaacagu A- 2586 VPusAfsggaCfuguucug UCACCAUCGACAGA 4837 12230 22823 ccuaL96 228238 UfcGfauggusgsa ACAGUCCUU 04.1 87.1 8.1 AD- A- 2138 cscsaucgAfcAfGfAfacaguc A- 2587 VPusAfsaggAfcuguucu CACCAUCGACAGAA 4838 12230 22823 cuuaL96 228239 GfuCfgauggsusg CAGUCCUUA 05.1 89.1 0.1 AD- A- 2139 csasucgaCfaGfAfAfcagucc A- 2588 VPusUfsaagGfacuguuc ACCAUCGACAGAAC 4839 12230 22823 uuaaL96 228239 UfgUfcgaugsgsu AGUCCUUAA 06.1 91.1 2.1 AD- A- 2140 asuscgacAfgAfAfCfaguccu A- 2589 VPusUfsuaaGfgacuguu CCAUCGACAGAACA 4840 12230 22823 uaaaL96 228239 CfuGfucgausgsg GUCCUUAAU 07.1 93.1 4.1 AD- A- 2141 uscsgacaGfaAfCfAfguccuu A- 2590 VPusAfsuuaAfggacugu CAUCGACAGAACAG 4841 12230 22823 aauaL96 228239 UfcUfgucgasusg UCCUUAAUC 08.1 95.1 6.1 AD- A- 2142 csgsacagAfaCfAfGfuccuua A- 2591 VPusGfsauuAfaggacug AUCGACAGAACAGU 4842 12230 22823 aucaL96 228239 UfuCfugucgsasu CCUUAAUCC 09.1 97.1 8.1 AD- A- 2143 gsascagaAfcAfGfUfccuuaa A- 2592 VPusGfsgauUfaaggacu UCGACAGAACAGUC 4843 12230 22823 uccaL96 228240 GfuUfcugucsgsa CUUAAUCCA 10.1 99.1 0.1 AD- A- 2144 ascsagaaCfaGfUfCfcuuaau A- 2593 VPusUfsggaUfuaaggac CGACAGAACAGUCC 4844 12230 22824 ccaaL96 228240 UfgUfucuguscsg UUAAUCCAG 11.1 01.1 2.1 AD- A- 2145 asgsaacaGfuCfCfUfuaaucc A- 2594 VPusUfscugGfauuaagg ACAGAACAGUCCUU 4845 12230 22824 agaaL96 228240 AfcUfguucusgsu AAUCCAGAA 12.1 03.1 4.1 AD- A- 2146 gsasacagUfcCfUfUfaaucca A- 2595 VPusUfsucuGfgauuaag CAGAACAGUCCUUA 4846 12230 22824 gaaaL96 228240 GfaCfuguucsusg AUCCAGAAA 13.1 05.1 6.1 AD- A- 2147 asascaguCfcUfUfAfauccag A- 2596 VPusUfsuucUfggauuaa AGAACAGUCCUUAA 4847 12230 22824 aaaaL96 228240 GfgAfcuguuscsu UCCAGAAAC 14.1 07.1 8.1 AD- A- 2148 ascsagucCfuUfAfAfuccaga A- 2597 VPusGfsuuuCfuggauua GAACAGUCCUUAAU 4848 12230 22824 aacaL96 228241 AfgGfacugususc CCAGAAACC 15.1 09.1 0.1 AD- A- 2149 csasguccUfuAfAfUfccagaa A- 2598 VPusGfsguuUfcuggauu AACAGUCCUUAAUC 4849 12230 22824 accaL96 228241 AfaGfgacugsusu CAGAAACCU 16.1 11.1 2.1 AD- A- 2150 asgsuccuUfaAfUfCfcagaaa A- 2599 VPusAfsgguUfucuggau ACAGUCCUUAAUCC 4850 12230 22824 ccuaL96 228241 UfaAfggacusgsu AGAAACCUG 17.1 13.1 4.1 AD- A- 2151 gsusccuuAfaUfCfCfagaaac A- 2600 VPusCfsaggUfuucugga CAGUCCUUAAUCCA 4851 12230 22824 cugaL96 228241 UfuAfaggacsusg GAAACCUGA 18.1 15.1 6.1 AD- A- 2152 uscscuuaAfuCfCfAfgaaacc A- 2601 VPusUfscagGfuuucugg AGUCCUUAAUCCAG 4852 12230 22824 ugaaL96 228241 AfuUfaaggascsu AAACCUGAA 19.1 17.1 8.1 AD- A- 2153 cscsuuaaUfcCfAfGfaaaccu A- 2602 VPusUfsucaGfguuucug GUCCUUAAUCCAGA 4853 12230 22824 gaaaL96 228242 GfaUfuaaggsasc AACCUGAAA 20.1 19.1 0.1 AD- A- 2154 csusuaauCfcAfGfAfaaccug A- 2603 VPusUfsuucAfgguuucu UCCUUAAUCCAGAA 4854 12230 22824 aaaaL96 228242 GfgAfuuaagsgsa ACCUGAAAU 21.1 21.1 2.1 AD- A- 2155 ususaaucCfaGfAfAfaccuga A- 2604 VPusAfsuuuCfagguuuc CCUUAAUCCAGAAA 4855 12230 22824 aauaL96 228242 UfgGfauuaasgsg CCUGAAAUG 22.1 23.1 4.1 AD- A- 2156 csasgaaaCfcUfGfAfaaugaa A- 2605 VPusUfsccuUfcauuucaG UCCAGAAACCUGAA 4856 12230 22824 ggaaL96 228242 fgUfuucugsgsa AUGAAGGAA 23.1 25.1 6.1 AD- A- 2157 asgsaaacCfuGfAfAfaugaag A- 2606 VPusUfsuccUfucauuuc CCAGAAACCUGAAA 4857 12230 22824 gaaaL96 228242 AfgGfuuucusgsg UGAAGGAAG 24.1 27.1 8.1 AD- A- 2158 gsasaaccUfgAfAfAfugaagg A- 2607 VPusCfsuucCfuucauuuC CAGAAACCUGAAAU 4858 12230 22824 aagaL96 228243 faGfguuucsusg GAAGGAAGA 25.1 29.1 0.1 AD- A- 2159 asasaccuGfaAfAfUfgaagga A- 2608 VPusUfscuuCfcuucauu AGAAACCUGAAAUG 4859 12230 22824 agaaL96 228243 UfcAfgguuuscsu AAGGAAGAG 26.1 31.1 2.1 AD- A- 2160 asasccugAfaAfUfGfaaggaa A- 2609 VPusCfsucuUfccuucauU GAAACCUGAAAUGA 4860 12230 22824 gagaL96 228243 fuCfagguususc AGGAAGAGG 27.1 33.1 4.1 AD- A- 2161 cscsugaaAfuGfAfAfggaaga A- 2610 VPusUfsccuCfuuccuucA AACCUGAAAUGAAG 4861 12230 22824 ggaaL96 228243 fuUfucaggsusu GAAGAGGAG 28.1 35.1 6.1 AD- A- 2162 asusgaagGfaAfGfAfggagac A- 2611 VPusAfsgagUfcuccucu AAAUGAAGGAAGA 4862 12230 22824 ucuaL96 228243 UfcCfuucaususu GGAGACUCUG 29.1 37.1 8.1 AD- A- 2163 uscsccucUfuGfGfAfauugga A- 2612 VPusGfsaauCfcaauuccA GGUCCCUCUUGGAA 4863 12230 22824 uucaL96 228244 faGfagggascsc UUGGAUUCG 30.1 39.1 0.1 AD- A- 2164 cscsucuuGfgAfAfUfuggauu A- 2613 VPusGfscgaAfuccaauuC UCCCUCUUGGAAUU 4864 12230 22824 cgcaL96 228244 fcAfagaggsgsa GGAUUCGCC 31.1 41.1 2.1 AD- A- 2165 csuscuugGfaAfUfUfggauuc A- 2614 VPusGfsgcgAfauccaauU CCCUCUUGGAAUUG 4865 12230 22824 gccaL96 228244 fcCfaagagsgsg GAUUCGCCA 32.1 43.1 4.1 AD- A- 2166 ususggaaUfuGfGfAfuucgcc A- 2615 VPusAfsaugGfcgaauccA UCUUGGAAUUGGAU 4866 12230 22824 auuaL96 228244 faUfuccaasgsa UCGCCAUUU 33.1 45.1 6.1 AD- A- 2167 usgsgaauUfgGfAfUfucgcca A- 2616 VPusAfsaauGfgcgaaucC CUUGGAAUUGGAUU 4867 12230 22824 uuuaL96 228244 faAfuuccasasg CGCCAUUUU 34.1 47.1 8.1 AD- A- 2168 gsgsaauuGfgAfUfUfcgccau A- 2617 VPusAfsaaaUfggcgaauC UUGGAAUUGGAUUC 4868 12230 22824 uuuaL96 228245 fcAfauuccsasa GCCAUUUUA 35.1 49.1 0.1 AD- A- 2169 gsasauugGfaUfUfCfgccauu A- 2618 VPusUfsaaaAfuggcgaaU UGGAAUUGGAUUCG 4869 12230 22824 uuaaL96 228245 fcCfaauucscsa CCAUUUUAU 36.1 51.1 2.1 AD- A- 2170 asasuuggAfuUfCfGfccauuu A- 2619 VPusAfsuaaAfauggcga GGAAUUGGAUUCGC 4870 12230 22824 uauaL96 228245 AfuCfcaauuscsc CAUUUUAUU 37.1 53.1 4.1 AD- A- 2171 asusuggaUfuCfGfCfcauuuu A- 2620 VPusAfsauaAfaauggcg GAAUUGGAUUCGCC 4871 12230 22824 auuaL96 228245 AfaUfccaaususc AUUUUAUUU 38.1 55.1 6.1 AD- A- 2172 ususggauUfcGfCfCfauuuua A- 2621 VPusAfsaauAfaaauggcG AAUUGGAUUCGCCA 4872 12230 22824 uuuaL96 228245 faAfuccaasusu UUUUAUUUU 39.1 57.1 8.1 AD- A- 2173 usgsgauuCfgCfCfAfuuuuau A- 2622 VPusAfsaaaUfaaaauggC AUUGGAUUCGCCAU 4873 12230 22824 uuuaL96 228246 fgAfauccasasu UUUAUUUUU 40.1 59.1 0.1 AD- A- 2174 gsgsauucGfcCfAfUfuuuauu A- 2623 VPusAfsaaaAfuaaaaugG UUGGAUUCGCCAUU 4874 12230 22824 uuuaL96 228246 fcGfaauccsasa UUAUUUUUC 41.1 61.1 2.1 AD- A- 2175 gsasuucgCfcAfUfUfuuauuu A- 2624 VPusGfsaaaAfauaaaauG UGGAUUCGCCAUUU 4875 12230 22824 uucaL96 228246 fgCfgaaucscsa UAUUUUUCU 42.1 63.1 4.1 AD- A- 2176 uscsgccaUfuUfUfAfuuuuuc A- 2625 VPusCfsaagAfaaaauaaA AUUCGCCAUUUUAU 4876 12230 22824 uugaL96 228246 faUfggcgasasu UUUUCUUGC 43.1 65.1 6.1 AD- A- 2177 csgsccauUfuUfAfUfuuuucu A- 2626 VPusGfscaaGfaaaaauaA UUCGCCAUUUUAUU 4877 12230 22824 ugcaL96 228246 faAfuggcgsasa UUUCUUGCU 44.1 67.1 8.1 AD- A- 2178 gscscauuUfuAfUfUfuuucuu A- 2627 VPusAfsgcaAfgaaaaauA UCGCCAUUUUAUUU 4878 12230 22824 gcuaL96 228247 faAfauggcsgsa UUCUUGCUG 45.1 69.1 0.1 AD- A- 2179 cscsauuuUfaUfUfUfuucuug A- 2628 VPusCfsagcAfagaaaaaU CGCCAUUUUAUUUU 4879 12230 22824 cugaL96 228247 faAfaauggscsg UCUUGCUGC 46.1 71.1 2.1 AD- A- 2180 csasuuuuAfuUfUfUfucuugc A- 2629 VPusGfscagCfaagaaaaA GCCAUUUUAUUUUU 4880 12230 22824 ugcaL96 228247 fuAfaaaugsgsc CUUGCUGCU 47.1 73.1 4.1 AD- A- 2181 asusuuuaUfuUfUfUfcuugcu A- 2630 VPusAfsgcaGfcaagaaaA CCAUUUUAUUUUUC 4881 12230 22824 gcuaL96 228247 faUfaaaausgsg UUGCUGCUA 48.1 75.1 6.1 AD- A- 2182 ususuuauUfuUfUfCfuugcu A- 2631 VPusUfsagcAfgcaagaaA CAUUUUAUUUUUCU 4882 12230 22824 gcuaaL96 228247 faAfuaaaasusg UGCUGCUAA 49.1 77.1 8.1 AD- A- 2183 ususucuuGfcUfGfCfuaaauc A- 2632 VPusGfsgugAfuuuagca UUUUUCUUGCUGCU 4883 12230 22824 accaL96 228248 GfcAfagaaasasa AAAUCACCG 50.1 79.1 0.1 AD- A- 2184 uscsaccgAfgCfCfCfggaaga A- 2633 VPusUfsaauCfuuccgggC AAUCACCGAGCCCG 4884 12230 22824 uuaaL96 228248 fuCfggugasusu GAAGAUUAG 51.1 81.1 2.1 AD- A- 2185 gscsccggAfaGfAfUfuagaga A- 2634 VPusAfsacuCfucuaaucU GAGCCCGGAAGAUU 4885 12230 22824 guuaL96 228248 fuCfcgggcsusc AGAGAGUUU 52.1 83.1 4.1 AD- A- 2186 cscscggaAfgAfUfUfagagag A- 2635 VPusAfsaacUfcucuaauC AGCCCGGAAGAUUA 4886 12230 22824 uuuaL96 228248 fuUfccgggscsu GAGAGUUUU 53.1 85.1 6.1 AD- A- 2187 csgsgaagAfuUfAfGfagaguu A- 2636 VPusUfsaaaAfcucucuaA CCCGGAAGAUUAGA 4887 12230 22824 uuaaL96 228248 fuCfuuccgsgsg GAGUUUUAU 54.1 87.1 8.1 AD- A- 2188 gsgsaagaUfuAfGfAfgaguuu A- 2637 VPusAfsuaaAfacucucuA CCGGAAGAUUAGAG 4888 12230 22824 uauaL96 228249 faUfcuuccsgsg AGUUUUAUU 55.1 89.1 0.1 AD- A- 2189 gsasagauUfaGfAfGfaguuuu A- 2638 VPusAfsauaAfaacucucU CGGAAGAUUAGAGA 4889 12230 22824 auuaL96 228249 faAfucuucscsg GUUUUAUUU 56.1 91.1 2.1 AD- A- 2190 asasgauuAfgAfGfAfguuuua A- 2639 VPusAfsaauAfaaacucuC GGAAGAUUAGAGA 4890 12230 22824 uuuaL96 228249 fuAfaucuuscsc GUUUUAUUUC 57.1 93.1 4.1 AD- A- 2191 asgsauuaGfaGfAfGfuuuuau A- 2640 VPusGfsaaaUfaaaacucU GAAGAUUAGAGAG 4891 12230 22824 uucaL96 228249 fcUfaaucususc UUUUAUUUCU 58.1 95.1 6.1 AD- A- 2192 asusuagaGfaGfUfUfuuauuu A- 2641 VPusCfsagaAfauaaaacU AGAUUAGAGAGUU 4892 12230 22824 cugaL96 228249 fcUfcuaauscsu UUAUUUCUGG 59.1 97.1 8.1 AD- A- 2193 ususagagAfgUfUfUfuauuuc A- 2642 VPusCfscagAfaauaaaaC GAUUAGAGAGUUU 4893 12230 22824 uggaL96 228250 fuCfucuaasusc UAUUUCUGGG 60.1 99.1 0.1 AD- A- 2194 usasgagaGfuUfUfUfauuucu A- 2643 VPusCfsccaGfaaauaaaA AUUAGAGAGUUUU 4894 12230 22825 gggaL96 228250 fcUfcucuasasu AUUUCUGGGA 61.1 01.1 2.1 AD- A- 2195 asgsagagUfuUfUfAfuuucug A- 2644 VPusUfscccAfgaaauaaA UUAGAGAGUUUUA 4895 12230 22825 ggaaL96 228250 faCfucucusasa UUUCUGGGAU 62.1 03.1 4.1 AD- A- 2196 gsasgaguUfuUfAfUfuucug A- 2645 VPusAfsuccCfagaaauaA UAGAGAGUUUUAU 4896 12230 22825 ggauaL96 228250 faAfcucucsusa UUCUGGGAUU 63.1 05.1 6.1 AD- A- 2197 asgsaguuUfuAfUfUfucugg A- 2646 VPusAfsaucCfcagaaauA AGAGAGUUUUAUU 4897 12230 22825 gauuaL96 228250 faAfacucuscsu UCUGGGAUUC 64.1 07.1 8.1 AD- A- 2198 gsasguuuUfaUfUfUfcuggga A- 2647 VPusGfsaauCfccagaaaU GAGAGUUUUAUUUC 4898 12230 22825 uucaL96 228251 faAfaacucsusc UGGGAUUCC 65.1 09.1 0.1 AD- A- 2199 asgsuuuuAfuUfUfCfuggga A- 2648 VPusGfsgaaUfcccagaaA AGAGUUUUAUUUCU 4899 12230 22825 uuccaL96 228251 fuAfaaacuscsu GGGAUUCCU 66.1 11.1 2.1 AD- A- 2200 gsusuuuaUfuUfCfUfgggau A- 2649 VPusAfsggaAfucccagaA GAGUUUUAUUUCUG 4900 12230 22825 uccuaL96 228251 faUfaaaacsusc GGAUUCCUG 67.1 13.1 4.1 AD- A- 2201 ususuuauUfuCfUfGfggauuc A- 2650 VPusCfsaggAfaucccagA AGUUUUAUUUCUGG 4901 12230 22825 cugaL96 228251 faAfuaaaascsu GAUUCCUGU 68.1 15.1 6.1 AD- A- 2202 ususuauuUfcUfGfGfgauucc A- 2651 VPusAfscagGfaaucccaG GUUUUAUUUCUGGG 4902 12230 22825 uguaL96 228251 faAfauaaasasc AUUCCUGUA 69.1 17.1 8.1 AD- A- 2203 ususauuuCfuGfGfGfauuccu A- 2652 VPusUfsacaGfgaaucccA UUUUAUUUCUGGGA 4903 12230 22825 guaaL96 228252 fgAfaauaasasa UUCCUGUAG 70.1 19.1 0.1 AD- A- 2204 usasuuucUfgGfGfAfuuccug A- 2653 VPusCfsuacAfggaauccC UUUAUUUCUGGGAU 4904 12230 22825 uagaL96 228252 faGfaaauasasa UCCUGUAGA 71.1 21.1 2.1 AD- A- 2205 asusuucuGfgGfAfUfuccugu A- 2654 VPusUfscuaCfaggaaucC UUAUUUCUGGGAUU 4905 12230 22825 agaaL96 228252 fcAfgaaausasa CCUGUAGAC 72.1 23.1 4.1 AD- A- 2206 ususucugGfgAfUfUfccugua A- 2655 VPusGfsucuAfcaggaauC UAUUUCUGGGAUUC 4906 12230 22825 gacaL96 228252 fcCfagaaasusa CUGUAGACA 73.1 25.1 6.1 AD- A- 2207 uscsugggAfuUfCfCfuguaga A- 2656 VPusGfsuguCfuacagga UUUCUGGGAUUCCU 4907 12230 22825 cacaL96 228252 AfuCfccagasasa GUAGACACA 74.1 27.1 8.1 AD- A- 2208 csusgggaUfuCfCfUfguagac A- 2657 VPusUfsgugUfcuacagg UUCUGGGAUUCCUG 4908 12230 22825 acaaL96 228253 AfaUfcccagsasa UAGACACAC 75.1 29.1 0.1 AD- A- 2209 usgsggauUfcCfUfGfuagaca A- 2658 VPusGfsuguGfucuacag UCUGGGAUUCCUGU 4909 12230 22825 cacaL96 228253 GfaAfucccasgsa AGACACACC 76.1 31.1 2.1 AD- A- 2210 gsgsgauuCfcUfGfUfagacac A- 2659 VPusGfsgugUfgucuaca CUGGGAUUCCUGUA 4910 12230 22825 accaL96 228253 GfgAfaucccsasg GACACACCC 77.1 33.1 4.1 AD- A- 2211 ascsacacCfcAfCfCfcacauac A- 2660 VPusAfsuguAfugugggu AGACACACCCACCC 4911 12230 22825 auaL96 228253 GfgGfuguguscsu ACAUACAUA 78.1 35.1 6.1 AD- A- 2212 ascsccacCfcAfCfAfuacauac A- 2661 VPusAfsuguAfuguaugu ACACCCACCCACAU 4912 12230 22825 auaL96 228253 GfgGfugggusgsu ACAUACAUU 79.1 37.1 8.1 AD- A- 2213 cscscaccCfaCfAfUfacauaca A- 2662 VPusAfsaugUfauguaug CACCCACCCACAUA 4913 12230 22825 uuaL96 228254 UfgGfgugggsusg CAUACAUUU 80.1 39.1 0.1 AD- A- 2214 cscsacccAfcAfUfAfcauaca A- 2663 VPusAfsaauGfuauguau ACCCACCCACAUAC 4914 12230 22825 uuuaL96 228254 GfuGfgguggsgsu AUACAUUUA 81.1 41.1 2.1 AD- A- 2215 csascccaCfaUfAfCfauacauu A- 2664 VPusUfsaaaUfguaugua CCCACCCACAUACA 4915 12230 22825 uaaL96 228254 UfgUfgggugsgsg UACAUUUAU 82.1 43.1 4.1 AD- A- 2216 cscsacauAfcAfUfAfcauuua A- 2665 VPusAfsuauAfaauguau ACCCACAUACAUAC 4916 12230 22825 uauaL96 228254 GfuAfuguggsgsu AUUUAUAUA 83.1 45.1 6.1 AD- A- 2217 csascauaCfaUfAfCfauuuau A- 2666 VPusUfsauaUfaaauguaU CCCACAUACAUACA 4917 12230 22825 auaaL96 228254 fgUfaugugsgsg UUUAUAUAU 84.1 47.1 8.1 AD- A- 2218 ususaaauUfaAfCfAfgugcua A- 2667 VPusCfsauuAfgcacugu UUUUAAAUUAACAG 4918 12230 22825 augaL96 228255 UfaAfuuuaasasa UGCUAAUGU 85.1 49.1 0.1 AD- A- 2219 usasaauuAfaCfAfGfugcuaa A- 2668 VPusAfscauUfagcacugU UUUAAAUUAACAGU 4919 12230 22825 uguaL96 228255 fuAfauuuasasa GCUAAUGUU 86.1 51.1 2.1 AD- A- 2220 asasauuaAfcAfGfUfgcuaau A- 2669 VPusAfsacaUfuagcacuG UUAAAUUAACAGUG 4920 12230 22825 guuaL96 228255 fuUfaauuusasa CUAAUGUUA 87.1 53.1 4.1 AD- A- 2221 asasuuaaCfaGfUfGfcuaaug A- 2670 VPusUfsaacAfuuagcacU UAAAUUAACAGUGC 4921 12230 22825 uuaaL96 228255 fgUfuaauususa UAAUGUUAU 88.1 55.1 6.1 AD- A- 2222 asusuaacAfgUfGfCfuaaugu A- 2671 VPusAfsuaaCfauuagcaC AAAUUAACAGUGCU 4922 12230 22825 uauaL96 228255 fuGfuuaaususu AAUGUUAUU 89.1 57.1 8.1 AD- A- 2223 ususaacaGfuGfCfUfaauguu A- 2672 VPusAfsauaAfcauuagcA AAUUAACAGUGCUA 4923 12230 22825 auuaL96 228256 fcUfguuaasusu AUGUUAUUG 90.1 59.1 0.1 AD- A- 2224 usasacagUfgCfUfAfauguua A- 2673 VPusCfsaauAfacauuagC AUUAACAGUGCUAA 4924 12230 22825 uugaL96 228256 faCfuguuasasu UGUUAUUGG 91.1 61.1 2.1 AD- A- 2225 asascaguGfcUfAfAfuguuau A- 2674 VPusCfscaaUfaacauuaG UUAACAGUGCUAAU 4925 12230 22825 uggaL96 228256 fcAfcuguusasa GUUAUUGGU 92.1 63.1 4.1 AD- A- 2226 ascsagugCfuAfAfUfguuauu A- 2675 VPusAfsccaAfuaacauuA UAACAGUGCUAAUG 4926 12230 22825 gguaL96 228256 fgCfacugususa UUAUUGGUG 93.1 65.1 6.1 AD- A- 2227 csasgugcUfaAfUfGfuuauug A- 2676 VPusCfsaccAfauaacauU AACAGUGCUAAUGU 4927 12230 22825 gugaL96 228256 faGfcacugsusu UAUUGGUGU 94.1 67.1 8.1 AD- A- 2228 gsusgcuaAfuGfUfUfauugg A- 2677 VPusGfsacaCfcaauaacA CAGUGCUAAUGUUA 4928 12230 22825 ugucaL96 228257 fuUfagcacsusg UUGGUGUCU 95.1 69.1 0.1 AD- A- 2229 usgscuaaUfgUfUfAfuuggu A- 2678 VPusAfsgacAfccaauaaC AGUGCUAAUGUUAU 4929 12230 22825 gucuaL96 228257 faUfuagcascsu UGGUGUCUU 96.1 71.1 2.1 AD- A- 2230 gscsuaauGfuUfAfUfuggug A- 2679 VPusAfsagaCfaccaauaA GUGCUAAUGUUAUU 4930 12230 22825 ucuuaL96 228257 fcAfuuagcsasc GGUGUCUUC 97.1 73.1 4.1 AD- A- 2231 csusaaugUfuAfUfUfgguguc A- 2680 VPusGfsaagAfcaccaauA UGCUAAUGUUAUUG 4931 12230 22825 uucaL96 228257 faCfauuagscsa GUGUCUUCA 98.1 75.1 6.1 AD- A- 2232 usasauguUfaUfUfGfgugucu A- 2681 VPusUfsgaaGfacaccaaU GCUAAUGUUAUUGG 4932 12230 22825 ucaaL96 228257 faAfcauuasgsc UGUCUUCAC 99.1 77.1 8.1 AD- A- 2233 asasuguuAfuUfGfGfugucu A- 2682 VPusGfsugaAfgacaccaA CUAAUGUUAUUGGU 4933 12231 22825 ucacaL96 228258 fuAfacauusasg GUCUUCACU 00.1 79.1 0.1 AD- A- 2234 asusguuaUfuGfGfUfgucuuc A- 2683 VPusAfsgugAfagacaccA UAAUGUUAUUGGU 4934 12231 22825 acuaL96 228258 faUfaacaususa GUCUUCACUG 01.1 81.1 2.1 AD- A- 2235 usgsuuauUfgGfUfGfucuuca A- 2684 VPusCfsaguGfaagacacC AAUGUUAUUGGUG 4935 12231 22825 cugaL96 228258 faAfuaacasusu UCUUCACUGG 02.1 83.1 4.1 AD- A- 2236 gsusuauuGfgUfGfUfcuucac A- 2685 VPusCfscagUfgaagacaC AUGUUAUUGGUGUC 4936 12231 22825 uggaL96 228258 fcAfauaacsasu UUCACUGGA 03.1 85.1 6.1 AD- A- 2237 ususauugGfuGfUfCfuucacu A- 2686 VPusUfsccaGfugaagacA UGUUAUUGGUGUCU 4937 12231 22825 ggaaL96 228258 fcCfaauaascsa UCACUGGAU 04.1 87.1 8.1 AD- A- 2238 usgsguguCfuUfCfAfcuggau A- 2687 VPusUfsacaUfccagugaA AUUGGUGUCUUCAC 4938 12231 22825 guaaL96 228259 fgAfcaccasasu UGGAUGUAU 05.1 89.1 0.1 AD- A- 2239 usgsucuuCfaCfUfGfgaugua A- 2688 VPusAfsaauAfcauccagU GGUGUCUUCACUGG 4939 12231 22825 uuuaL96 228259 fgAfagacascsc AUGUAUUUG 06.1 91.1 2.1 AD- A- 2240 csascuggAfuGfUfAfuuugac A- 2689 VPusGfscagUfcaaauacA UUCACUGGAUGUAU 4940 12231 22825 ugcaL96 228259 fuCfcagugsasa UUGACUGCU 07.1 93.1 4.1 AD- A- 2241 ascsuggaUfgUfAfUfuugacu A- 2690 VPusAfsgcaGfucaaauaC UCACUGGAUGUAUU 4941 12231 22825 gcuaL96 228259 faUfccagusgsa UGACUGCUG 08.1 95.1 6.1 AD- A- 2242 usgsgaugUfaUfUfUfgacugc A- 2691 VPusAfscagCfagucaaaU ACUGGAUGUAUUUG 4942 12231 22825 uguaL96 228259 faCfauccasgsu ACUGCUGUG 09.1 97.1 8.1 AD- A- 2243 usasuuugAfcUfGfCfugugga A- 2692 VPusAfsaguCfcacagcaG UGUAUUUGACUGCU 4943 12231 22825 cuuaL96 228260 fuCfaaauascsa GUGGACUUG 10.1 99.1 0.1 AD- A- 2244 ususugacUfgCfUfGfuggacu A- 2693 VPusUfscaaGfuccacagC UAUUUGACUGCUGU 4944 12231 22826 ugaaL96 228260 faGfucaaasusa GGACUUGAG 11.1 01.1 2.1 AD- A- 2245 gscsugugGfaCfUfUfgaguug A- 2694 VPusUfscccAfacucaagU CUGCUGUGGACUUG 4945 12231 22826 ggaaL96 228260 fcCfacagcsasg AGUUGGGAG 12.1 03.1 4.1 AD- A- 2246 uscsccacUfcAfGfAfuccuga A- 2695 VPusCfsuguCfaggaucu GUUCCCACUCAGAU 4946 12231 22826 cagaL96 228260 GfaGfugggasasc CCUGACAGG 13.1 05.1 6.1 AD- A- 2247 cscscacuCfaGfAfUfccugac A- 2696 VPusCfscugUfcaggaucU UUCCCACUCAGAUC 4947 12231 22826 aggaL96 228260 fgAfgugggsasa CUGACAGGG 14.1 07.1 8.1 AD- A- 2248 gsgsaggaGfaUfGfAfgagacu A- 2697 VPusCfsagaGfucucucaU GAGGAGGAGAUGA 4948 12231 22826 cugaL96 228261 fcUfccuccsusc GAGACUCUGG 15.1 09.1 0.1 AD- A- 2249 gsasggagAfuGfAfGfagacuc A- 2698 VPusCfscagAfgucucucA AGGAGGAGAUGAG 4949 12231 22826 uggaL96 228261 fuCfuccucscsu AGACUCUGGC 16.1 11.1 2.1 AD- A- 2250 gsgsagauGfaGfAfGfacucug A- 2699 VPusUfsgccAfgagucuc GAGGAGAUGAGAG 4950 12231 22826 gcaaL96 228261 UfcAfucuccsusc ACUCUGGCAU 17.1 13.1 4.1 AD- A- 2251 gsasgacuCfuGfGfCfaugauc A- 2700 VPusAfsaagAfucaugccA GAGAGACUCUGGCA 4951 12231 22826 uuuaL96 228261 fgAfgucucsusc UGAUCUUUU 18.1 15.1 6.1 AD- A- 2252 asgsacucUfgGfCfAfugaucu A- 2701 VPusAfsaaaGfaucaugcC AGAGACUCUGGCAU 4952 12231 22826 uuuaL96 228261 faGfagucuscsu GAUCUUUUU 19.1 17.1 8.1 AD- A- 2253 ususuuggGfaAfCfAfccgaca A- 2702 VPusGfsuuuGfucggugu AGUUUUGGGAACAC 4953 12231 22826 aacaL96 228262 UfcCfcaaaascsu CGACAAACC 20.1 19.1 0.1 AD- A- 2254 gsasgcuuCfaGfGfAfcauugc A- 2703 VPusAfscagCfaauguccU GGGAGCUUCAGGAC 4954 12231 22826 uguaL96 228262 fgAfagcucscsc AUUGCUGUG 21.1 21.1 2.1 AD- A- 2255 gscsuucaGfgAfCfAfuugcug A- 2704 VPusGfscacAfgcaauguC GAGCUUCAGGACAU 4955 12231 22826 ugcaL96 228262 fcUfgaagcsusc UGCUGUGCU 22.1 23.1 4.1 AD- A- 2256 csusucagGfaCfAfUfugcugu A- 2705 VPusAfsgcaCfagcaaugU AGCUUCAGGACAUU 4956 12231 22826 gcuaL96 228262 fcCfugaagscsu GCUGUGCUU 23.1 25.1 6.1 AD- A- 2257 ususcaggAfcAfUfUfgcugug A- 2706 VPusAfsagcAfcagcaauG GCUUCAGGACAUUG 4957 12231 22826 cuuaL96 228262 fuCfcugaasgsc CUGUGCUUU 24.1 27.1 8.1 AD- A- 2258 uscsaggaCfaUfUfGfcugugc A- 2707 VPusAfsaagCfacagcaaU CUUCAGGACAUUGC 4958 12231 22826 uuuaL96 228263 fgUfccugasasg UGUGCUUUG 25.1 29.1 0.1 AD- A- 2259 csasggacAfuUfGfCfugugcu A- 2708 VPusCfsaaaGfcacagcaA UUCAGGACAUUGCU 4959 12231 22826 uugaL96 228263 fuGfuccugsasa GUGCUUUGG 26.1 31.1 2.1 AD- A- 2260 csgscuuaCfuCfUfCfaccugc A- 2709 VPusGfsaagCfaggugag UUCGCUUACUCUCA 4960 12231 22826 uucaL96 228263 AfgUfaagcgsasa CCUGCUUCU 27.1 33.1 4.1 AD- A- 2261 gscsuuacUfcUfCfAfccugcu A- 2710 VPusAfsgaaGfcagguga UCGCUUACUCUCAC 4961 12231 22826 ucuaL96 228263 GfaGfuaagcsgsa CUGCUUCUG 28.1 35.1 6.1 AD- A- 2262 ususacucUfcAfCfCfugcuuc A- 2711 VPusUfscagAfagcaggu GCUUACUCUCACCU 4962 12231 22826 ugaaL96 228263 GfaGfaguaasgsc GCUUCUGAG 29.1 37.1 8.1 AD- A- 2263 csuscucaCfcUfGfCfuucuga A- 2712 VPusAfsacuCfagaagcaG UACUCUCACCUGCU 4963 12231 22826 guuaL96 228264 fgUfgagagsusa UCUGAGUUG 30.1 39.1 0.1 AD- A- 2264 uscsucacCfuGfCfUfucugag A- 2713 VPusCfsaacUfcagaagcA ACUCUCACCUGCUU 4964 12231 22826 uugaL96 228264 fgGfugagasgsu CUGAGUUGC 31.1 41.1 2.1 AD- A- 2265 csuscaccUfgCfUfUfcugagu A- 2714 VPusGfscaaCfucagaagC CUCUCACCUGCUUC 4965 12231 22826 ugcaL96 228264 faGfgugagsasg UGAGUUGCC 32.1 43.1 4.1 AD- A- 2266 uscsaccuGfcUfUfCfugaguu A- 2715 VPusGfsgcaAfcucagaaG UCUCACCUGCUUCU 4966 12231 22826 gccaL96 228264 fcAfggugasgsa GAGUUGCCC 33.1 45.1 6.1 AD- A- 2267 csasccugCfuUfCfUfgaguug A- 2716 VPusGfsggcAfacucagaA CUCACCUGCUUCUG 4967 12231 22826 cccaL96 228264 fgCfaggugsasg AGUUGCCCA 34.1 47.1 8.1 AD- A- 2268 ascscugcUfuCfUfGfaguugc A- 2717 VPusUfsgggCfaacucagA UCACCUGCUUCUGA 4968 12231 22826 ccaaL96 228265 faGfcaggusgsa GUUGCCCAG 35.1 49.1 0.1 AD- A- 2269 cscsugcuUfcUfGfAfguugcc A- 2718 VPusCfsuggGfcaacucaG CACCUGCUUCUGAG 4969 12231 22826 cagaL96 228265 faAfgcaggsusg UUGCCCAGG 36.1 51.1 2.1 AD- A- 2270 csgsgcgaAfgAfGfAfagagac A- 2719 VPusUfsgugUfcucuucu CCCGGCGAAGAGAA 4970 12231 22826 acaaL96 228265 CfuUfcgccgsgsg GAGACACAU 37.1 53.1 4.1 AD- A- 2271 gsgscgaaGfaGfAfAfgagaca A- 2720 VPusAfsuguGfucucuuc CCGGCGAAGAGAAG 4971 12231 22826 cauaL96 228265 UfcUfucgccsgsg AGACACAUU 38.1 55.1 6.1 AD- A- 2272 gscsgaagAfgAfAfGfagacac A- 2721 VPusAfsaugUfgucucuu CGGCGAAGAGAAGA 4972 12231 22826 auuaL96 228265 CfuCfuucgcscsg GACACAUUG 39.1 57.1 8.1 AD- A- 2273 csgsaagaGfaAfGfAfgacaca A- 2722 VPusCfsaauGfugucucu GGCGAAGAGAAGAG 4973 12231 22826 uugaL96 228266 UfcUfcuucgscsc ACACAUUGU 40.1 59.1 0.1 AD- A- 2274 gsasagagAfaGfAfGfacacau A- 2723 VPusAfscaaUfgugucuc GCGAAGAGAAGAGA 4974 12231 22826 uguaL96 228266 UfuCfucuucsgsc CACAUUGUU 41.1 61.1 2.1 AD- A- 2275 asasgagaAfgAfGfAfcacauu A- 2724 VPusAfsacaAfugugucu CGAAGAGAAGAGAC 4975 12231 22826 guuaL96 228266 CfuUfcucuuscsg ACAUUGUUG 42.1 63.1 4.1 AD- A- 2276 asgsagaaGfaGfAfCfacauug A- 2725 VPusCfsaacAfaugugucU GAAGAGAAGAGACA 4976 12231 22826 uugaL96 228266 fcUfucucususc CAUUGUUGG 43.1 65.1 6.1 AD- A- 2277 gsasgaagAfgAfCfAfcauugu A- 2726 VPusCfscaaCfaauguguC AAGAGAAGAGACAC 4977 12231 22826 uggaL96 228266 fuCfuucucsusu AUUGUUGGA 44.1 67.1 8.1 AD- A- 2278 asgsaagaGfaCfAfCfauuguu A- 2727 VPusUfsccaAfcaaugugU AGAGAAGAGACACA 4978 12231 22826 ggaaL96 228267 fcUfcuucuscsu UUGUUGGAA 45.1 69.1 0.1 AD- A- 2279 gsasagagAfcAfCfAfuuguug A- 2728 VPusUfsuccAfacaauguG GAGAAGAGACACAU 4979 12231 22826 gaaaL96 228267 fuCfucuucsusc UGUUGGAAG 46.1 71.1 2.1 AD- A- 2280 asasgagaCfaCfAfUfuguugg A- 2729 VPusCfsuucCfaacaaugU AGAAGAGACACAUU 4980 12231 22826 aagaL96 228267 fgUfcucuuscsu GUUGGAAGA 47.1 73.1 4.1 AD- A- 2281 asgsagacAfcAfUfUfguugga A- 2730 VPusUfscuuCfcaacaauG GAAGAGACACAUUG 4981 12231 22826 agaaL96 228267 fuGfucucususc UUGGAAGAA 48.1 75.1 6.1 AD- A- 2282 gsasgacaCfaUfUfGfuuggaa A- 2731 VPusUfsucuUfccaacaaU AAGAGACACAUUGU 4982 12231 22826 gaaaL96 228267 fgUfgucucsusu UGGAAGAAG 49.1 77.1 8.1 AD- A- 2283 asgsacacAfuUfGfUfuggaag A- 2732 VPusCfsuucUfuccaacaA AGAGACACAUUGUU 4983 12231 22826 aagaL96 228268 fuGfugucuscsu GGAAGAAGC 50.1 79.1 0.1 AD- A- 2284 gsascacaUfuGfUfUfggaaga A- 2733 VPusGfscuuCfuuccaacA GAGACACAUUGUUG 4984 12231 22826 agcaL96 228268 faUfgugucsusc GAAGAAGCA 51.1 81.1 2.1 AD- A- 2285 ascsacauUfgUfUfGfgaagaa A- 2734 VPusUfsgcuUfcuuccaaC AGACACAUUGUUGG 4985 12231 22826 gcaaL96 228268 faAfugugusesu AAGAAGCAG 52.1 83.1 4.1 AD- A- 2286 csascauuGfuUfGfGfaagaag A- 2735 VPusCfsugcUfucuuccaA GACACAUUGUUGGA 4986 12231 22826 cagaL96 228268 fcAfaugugsusc AGAAGCAGC 53.1 85.1 6.1 AD- A- 2287 usasugucCfuCfAfCfaccauu A- 2736 VPusUfsucaAfuggugug CCUAUGUCCUCACA 4987 12231 22826 gaaaL96 228268 AfgGfacauasgsg CCAUUGAAA 54.1 87.1 8.1 AD- A- 2288 uscscucaCfaCfCfAfuugaaa A- 2737 VPusUfsgguUfucaaugg UGUCCUCACACCAU 4988 12231 22826 ccaaL96 228269 UfgUfgaggascsa UGAAACCAC 55.1 89.1 0.1 AD- A- 2289 cscsucacAfcCfAfUfugaaac A- 2738 VPusGfsuggUfuucaaug GUCCUCACACCAUU 4989 12231 22826 cacaL96 228269 GfuGfugaggsasc GAAACCACU 56.1 91.1 2.1 AD- A- 2290 csuscacaCfcAfUfUfgaaacca A- 2739 VPusAfsgugGfuuucaau UCCUCACACCAUUG 4990 12231 22826 cuaL96 228269 GfgUfgugagsgsa AAACCACUA 57.1 93.1 4.1 AD- A- 2291 uscsacacCfaUfUfGfaaaccac A- 2740 VPusUfsaguGfguuucaa CCUCACACCAUUGA 4991 12231 22826 uaaL96 228269 UfgGfugugasgsg AACCACUAG 58.1 95.1 6.1 AD- A- 2292 csascaccAfuUfGfAfaaccac A- 2741 VPusCfsuagUfgguuuca CUCACACCAUUGAA 4992 12231 22826 uagaL96 228269 AfuGfgugugsasg ACCACUAGU 59.1 97.1 8.1 AD- A- 2293 cscsauugAfaAfCfCfacuagu A- 2742 VPusAfsgaaCfuaguggu CACCAUUGAAACCA 4993 12231 22826 ucuaL96 228270 UfuCfaauggsusg CUAGUUCUG 60.1 99.1 0.1 AD- A- 2294 csasuugaAfaCfCfAfcuaguu A- 2743 VPusCfsagaAfcuagugg ACCAUUGAAACCAC 4994 12231 22827 cugaL96 228270 UfuUfcaaugsgsu UAGUUCUGU 61.1 01.1 2.1 AD- A- 2295 asusugaaAfcCfAfCfuaguuc A- 2744 VPusAfscagAfacuagug CCAUUGAAACCACU 4995 12231 22827 uguaL96 228270 GfuUfucaausgsg AGUUCUGUC 62.1 03.1 4.1 AD- A- 2296 ususgaaaCfAfCfUfaguucu A- 2745 VPusGfsacaGfaacuaguG CAUUGAAACCACUA 4996 12231 22827 gucaL96 228270 fgUfuucaasusg GUUCUGUCC 63.1 05.1 6.1 AD- A- 2297 usgsaaacCfaCfUfAfguucug A- 2746 VPusGfsgacAfgaacuagU AUUGAAACCACUAG 4997 12231 22827 uccaL96 228270 fgGfuuucasasu UUCUGUCCC 64.1 07.1 8.1 AD- A- 2298 gsasccugGfuUfGfUfgugug A- 2747 VPusCfsacaCfacacacaAf GAGACCUGGUUGUG 4998 12231 22827 ugugaL96 228271 cCfaggucsusc UGUGUGUGA 65.1 09.1 0.1 AD- A- 2299 ascscuggUfuGfUfGfugugu A- 2748 VPusUfscacAfcacacacA AGACCUGGUUGUGU 4999 12231 22827 gugaaL96 228271 faCfcagguscsu GUGUGUGAG 66.1 11.1 2.1 AD- A- 2300 cscsugguUfgUfGfUfgugug A- 2749 VPusCfsucaCfacacacaCf GACCUGGUUGUGUG 5000 12231 22827 ugagaL96 228271 aAfccaggsusc UGUGUGAGU 67.1 13.1 4.1 AD- A- 2301 csusgguuGfuGfUfGfugugu A- 2750 VPusAfscucAfcacacacA ACCUGGUUGUGUGU 5001 12231 22827 gaguaL96 228271 fcAfaccagsgsu GUGUGAGUG 68.1 15.1 6.1 AD- A- 2302 usgsguugUfgUfGfUfgugug A- 2751 VPusCfsacuCfacacacaCf CCUGGUUGUGUGUG 5002 12231 22827 agugaL96 228271 aCfaaccasgsg UGUGAGUGG 69.1 17.1 8.1 AD- A- 2303 gsgsuuguGfuGfUfGfuguga A- 2752 VPusCfscacUfcacacacA CUGGUUGUGUGUGU 1914 12231 22827 guggaL96 228272 fcAfcaaccsasg GUGAGUGGU 70.1 19.1 0.1 AD- A- 2304 gsusguguGfuGfUfGfagugg A- 2753 VPusUfscaaCfcacucacA UUGUGUGUGUGUG 1915 12231 22827 uugaaL96 228272 fcAfcacacsasa AGUGGUUGAC 71.1 21.1 2.1 AD- A- 2305 usgsugugUfgUfGfAfguggu A- 2754 VPusGfsucaAfccacucaC UGUGUGUGUGUGA 1916 12231 22827 ugacaL96 228272 faCfacacascsa GUGGUUGACC 72.1 23.1 4.1 AD- A- 2306 usgsugugUfgAfGfUfgguug A- 2755 VPusAfsgguCfaaccacuC UGUGUGUGUGAGU 1917 12231 22827 accuaL96 228272 faCfacacascsa GGUUGACCUU 73.1 25.1 6.1 AD- A- 2307 usgsugugAfgUfGfGfuugac A- 2756 VPusGfsaagGfucaaccaC UGUGUGUGAGUGG 1918 12231 22827 cuucaL96 228272 fuCfacacascsa UUGACCUUCC 74.1 27.1 8.1 AD- A- 2308 gsusgugaGfuGfGfUfugaccu A- 2757 VPusGfsgaaGfgucaaccA GUGUGUGAGUGGU 1919 12231 22827 uccaL96 228273 fcUfcacacsasc UGACCUUCCU 75.1 29.1 0.1 AD- A- 2309 usgsugagUfgGfUfUfgaccuu A- 2758 VPusAfsggaAfggucaacC UGUGUGAGUGGUU 1920 12231 22827 ccuaL96 228273 faCfucacascsa GACCUUCCUC 76.1 31.1 2.1 AD- A- 2310 usgsagugGfuUfGfAfccuucc A- 2759 VPusGfsgagGfaagguca UGUGAGUGGUUGAC 1921 12231 22827 uccaL96 228273 AfcCfacucascsa CUUCCUCCA 77.1 33.1 4.1 AD- A- 2311 gsusgguuGfaCfCfUfuccucc A- 2760 VPusGfsaugGfaggaagg GAGUGGUUGACCUU 1922 12231 22827 aucaL96 228273 UfcAfaccacsusc CCUCCAUCC 78.1 35.1 6.1 AD- A- 2312 ususguggAfgGfCfAfgagaaa A- 2761 VPusUfscuuUfucucugc CAUUGUGGAGGCAG 1923 12231 22827 agaaL96 228273 CfuCfcacaasusg AGAAAAGAG 79.1 37.1 8.1 AD- A- 2313 usgsuggaGfgCfAfGfagaaaa A- 2762 VPusCfsucuUfuucucug AUUGUGGAGGCAGA 1924 12231 22827 gagaL96 228274 CfcUfccacasasu GAAAAGAGA 80.1 39.1 0.1 AD- A- 2314 gsusggagGfcAfGfAfgaaaag A- 2763 VPusUfscucUfuuucucu UUGUGGAGGCAGAG 1925 12231 22827 agaaL96 228274 GfcCfuccacsasa AAAAGAGAA 81.1 41.1 2.1 AD- A- 2315 usgsgaggCfaGfAfGfaaaaga A- 2764 VPusUfsucuCfuuuucuc UGUGGAGGCAGAGA 1926 12231 22827 gaaaL96 228274 UfgCfcuccascsa AAAGAGAAA 82.1 43.1 4.1 AD- A- 2316 gsgsaggcAfgAfGfAfaaagag A- 2765 VPusUfsuucUfcuuuucu GUGGAGGCAGAGAA 1927 12231 22827 aaaaL96 228274 CfuGfccuccsasc AAGAGAAAG 83.1 45.1 6.1 AD- A- 2317 gsasggcaGfaGfAfAfaagaga A- 2766 VPusCfsuuuCfucuuuuc UGGAGGCAGAGAAA 1928 12231 22827 aagaL96 228274 UfcUfgccucscsa AGAGAAAGU 84.1 47.1 8.1 AD- A- 2318 asgsgcagAfgAfAfAfagagaa A- 2767 VPusAfscuuUfcucuuuu GGAGGCAGAGAAAA 1929 12231 22827 aguaL96 228275 CfuCfugccuscsc GAGAAAGUG 85.1 49.1 0.1 AD- A- 2319 gsgscagaGfaAfAfAfgagaaa A- 2768 VPusCfsacuUfucucuuu GAGGCAGAGAAAAG 1930 12231 22827 gugaL96 228275 UfcUfcugccsusc AGAAAGUGU 86.1 51.1 2.1 AD- A- 2320 gscsagagAfaAfAfGfagaaag A- 2769 VPusAfscacUfuucucuu AGGCAGAGAAAAGA 1931 12231 22827 uguaL96 228275 UfuCfucugcscsu GAAAGUGUU 87.1 53.1 4.1 AD- A- 2321 csasgagaAfaAfGfAfgaaagu A- 2770 VPusAfsacaCfuuucucuU GGCAGAGAAAAGAG 1932 12231 22827 guuaL96 228275 fuUfcucugscsc AAAGUGUUU 88.1 55.1 6.1 AD- A- 2322 asgsagaaAfaGfAfGfaaagug A- 2771 VPusAfsaacAfcuuucucU GCAGAGAAAAGAGA 1933 12231 22827 uuuaL96 228275 fuUfucucusgsc AAGUGUUUU 89.1 57.1 8.1 AD- A- 2323 gsasgaaaAfgAfGfAfaagugu A- 2772 VPusAfsaaaCfacuuucuC CAGAGAAAAGAGAA 1934 12231 22827 uuuaL96 228276 fuUfuucucsusg AGUGUUUUA 90.1 59.1 0.1 AD- A- 2324 asgsaaaaGfaGfAfAfaguguu A- 2773 VPusUfsaaaAfcacuuucU AGAGAAAAGAGAA 1935 12231 22827 uuaaL96 228276 fcUfuuucuscsu AGUGUUUUAU 91.1 61.1 2.1 AD- A- 2325 gsasaaagAfgAfAfAfguguuu A- 2774 VPusAfsuaaAfacacuuuC GAGAAAAGAGAAA 1936 12231 22827 uauaL96 228276 fuCfuuuucsusc GUGUUUUAUA 92.1 63.1 4.1 AD- A- 2326 asasaagaGfaAfAfGfuguuuu A- 2775 VPusUfsauaAfaacacuuU AGAAAAGAGAAAG 1937 12231 22827 auaaL96 228276 fcUfcuuuuscsu UGUUUUAUAU 93.1 65.1 6.1 AD- A- 2327 asasagagAfaAfGfUfguuuua A- 2776 VPusAfsuauAfaaacacuU GAAAAGAGAAAGU 1938 12231 22827 uauaL96 228276 fuCfucuuususc GUUUUAUAUA 94.1 67.1 8.1 AD- A- 2328 asasgagaAfaGfUfGfuuuuau A- 2777 VPusUfsauaUfaaaacacU AAAAGAGAAAGUG 1939 12231 22827 auaaL96 228277 fuUfcucuususu UUUUAUAUAC 95.1 69.1 0.1 AD- A- 2329 asgsagaaAfgUfGfUfuuuaua A- 2778 VPusGfsuauAfuaaaacaC AAAGAGAAAGUGU 1940 12231 22827 uacaL96 228277 fuUfucucususu UUUAUAUACG 96.1 71.1 2.1 AD- A- 2330 gsasgaaaGfuGfUfUfuuauau A- 2779 VPusCfsguaUfauaaaacA AAGAGAAAGUGUU 1941 12231 22827 acgaL96 228277 fcUfuucucsusu UUAUAUACGG 97.1 73.1 4.1 AD- A- 2331 asgsaaagUfgUfUfUfuauaua A- 2780 VPusCfscguAfuauaaaaC AGAGAAAGUGUUU 1942 12231 22827 cggaL96 228277 faCfuuucuscsu UAUAUACGGU 98.1 75.1 6.1 AD- A- 2332 asasagugUfuUfUfAfuauacg A- 2781 VPusUfsaccGfuauauaaA AGAAAGUGUUUUA 1943 12231 22827 guaaL96 228277 faCfacuuuscsu UAUACGGUAC 99.1 77.1 8.1 AD- A- 2333 asasguguUfuUfAfUfauacgg A- 2782 VPusGfsuacCfguauauaA GAAAGUGUUUUAU 1944 12232 22827 uacaL96 228278 faAfcacuususc AUACGGUACU 00.1 79.1 0.1 AD- A- 2334 asgsuguuUfuAfUfAfuacgg A- 2783 VPusAfsguaCfcguauau AAAGUGUUUUAUA 1945 12232 22827 uacuaL96 228278 AfaAfacacususu UACGGUACUU 01.1 81.1 2.1 AD- A- 2335 gsusguuuUfaUfAfUfacggua A- 2784 VPusAfsaguAfccguaua AAGUGUUUUAUAU 1946 12232 22827 cuuaL96 228278 UfaAfaacacsusu ACGGUACUUA 02.1 83.1 4.1 AD- A- 2336 usgsuuuuAfuAfUfAfcggua A- 2785 VPusUfsaagUfaccguauA AGUGUUUUAUAUAC 1947 12232 22827 cuuaaL96 228278 fuAfaaacascsu GGUACUUAU 03.1 85.1 6.1 AD- A- 2337 gsusuuuaUfaUfAfCfgguacu A- 2786 VPusAfsuaaGfuaccguaU GUGUUUUAUAUACG 1948 12232 22827 uauaL96 228278 faUfaaaacsasc GUACUUAUU 04.1 87.1 8.1 AD- A- 2338 ususuuauAfuAfCfGfguacuu A- 2787 VPusAfsauaAfguaccgu UGUUUUAUAUACGG 1949 12232 22827 auuaL96 228279 AfuAfuaaaascsa UACUUAUUU 05.1 89.1 0.1 AD- A- 2339 ususuauaUfaCfGfGfuacuua A- 2788 VPusAfsaauAfaguaccgU GUUUUAUAUACGGU 1950 12232 22827 uuuaL96 228279 faUfauaaasasc ACUUAUUUA 06.1 91.1 2.1 AD- A- 2340 ususauauAfcGfGfUfacuuau A- 2789 VPusUfsaaaUfaaguaccG UUUUAUAUACGGUA 5040 12232 22827 uuaaL96 228279 fuAfuauaasasa CUUAUUUAA 07.1 93.1 4.1 AD- A- 2341 usasuauaCfgGfUfAfcuuauu A- 2790 VPusUfsuaaAfuaaguacC UUUAUAUACGGUAC 5041 12232 22827 uaaaL96 228279 fgUfauauasasa UUAUUUAAU 08.1 95.1 6.1 AD- A- 2342 gsusacuuAfuUfUfAfauaucc A- 2791 VPusAfsaggGfauauuaa CGGUACUUAUUUAA 5042 12232 22827 cuuaL96 228279 AfuAfaguacscsg UAUCCCUUU 09.1 97.1 8.1 AD- A- 2343 usascuuaUfuUfAfAfuauccc A- 2792 VPusAfsaagGfgauauua GGUACUUAUUUAAU 5043 12232 22827 uuuaL96 228280 AfaUfaaguascsc AUCCCUUUU 10.1 99.1 0.1 AD- A- 2344 ususaugaGfaUfGfUfaucuuu A- 2793 VPusGfscaaAfagauacaU AUUUAUGAGAUGU 5044 12232 22828 ugcaL96 228280 fcUfcauaasasu AUCUUUUGCU 11.1 01.1 2.1 AD- A- 2345 usasugagAfuGfUfAfucuuu A- 2794 VPusAfsgcaAfaagauacA UUUAUGAGAUGUA 5045 12232 22828 ugcuaL96 228280 fuCfucauasasa UCUUUUGCUC 12.1 03.1 4.1 AD- A- 2346 asusgagaUfgUfAfUfcuuuug A- 2795 VPusGfsagcAfaaagauaC UUAUGAGAUGUAUC 5046 12232 22828 cucaL96 228280 faUfcucausasa UUUUGCUCU 13.1 05.1 6.1 AD- A- 2347 usgsagauGfuAfUfCfuuuugc A- 2796 VPusAfsgagCfaaaagauA UAUGAGAUGUAUCU 5047 12232 22828 ucuaL96 228280 fcAfucucasusa UUUGCUCUC 14.1 07.1 8.1 AD- A- 2348 gsasgaugUfaUfCfUfuuugcu A- 2797 VPusGfsagaGfcaaaagaU AUGAGAUGUAUCUU 5048 12232 22828 cucaL96 228281 faCfaucucsasu UUGCUCUCU 15.1 09.1 0.1 AD- A- 2349 asgsauguAfuCfUfUfuugcuc A- 2798 VPusAfsgagAfgcaaaagA UGAGAUGUAUCUUU 5049 12232 22828 ucuaL96 228281 fuAfcaucuscsa UGCUCUCUC 16.1 11.1 2.1 AD- A- 2350 gsasuguaUfcUfUfUfugcucu A- 2799 VPusGfsagaGfagcaaaaG GAGAUGUAUCUUUU 5050 12232 22828 cucaL96 228281 faUfacaucsusc GCUCUCUCU 17.1 13.1 4.1 AD- A- 2351 asusguauCfuUfUfUfgcucuc A- 2800 VPusAfsgagAfgagcaaaA AGAUGUAUCUUUUG 5051 12232 22828 ucuaL96 228281 fgAfuacauscsu CUCUCUCUU 18.1 15.1 6.1 AD- A- 2352 gscsucucUfcUfUfGfcucucu A- 2801 VPusAfsuaaGfagagcaaG UUGCUCUCUCUUGC 5052 12232 22828 uauaL96 228281 faGfagagcsasa UCUCUUAUU 19.1 17.1 8.1 AD- A- 2353 csuscucuCfuUfGfCfucucuu A- 2802 VPusAfsauaAfgagagcaA UGCUCUCUCUUGCU 5053 12232 22828 auuaL96 228282 fgAfgagagscsa CUCUUAUUU 20.1 19.1 0.1 AD- A- 2354 uscsucucUfuGfCfUfcucuua A- 2803 VPusAfsaauAfagagagcA GCUCUCUCUUGCUC 5054 12232 22828 uuuaL96 228282 faGfagagasgsc UCUUAUUUG 21.1 21.1 2.1 AD- A- 2355 csuscucuUfgCfUfCfucuuau A- 2804 VPusCfsaaaUfaagagagC CUCUCUCUUGCUCU 5055 12232 22828 uugaL96 228282 faAfgagagsasg CUUAUUUGU 22.1 23.1 4.1 AD- A- 2356 uscsucuuGfcUfCfUfcuuauu A- 2805 VPusAfscaaAfuaagagaG UCUCUCUUGCUCUC 5056 12232 22828 uguaL96 228282 fcAfagagasgsa UUAUUUGUA 23.1 25.1 6.1 AD- A- 2357 csuscuugCfuCfUfCfuuauuu A- 2806 VPusUfsacaAfauaagagA CUCUCUUGCUCUCU 5057 12232 22828 guaaL96 228282 fgCfaagagsasg UAUUUGUAC 24.1 27.1 8.1 AD- A- 2358 uscsuugcUfcUfCfUfuauuug A- 2807 VPusGfsuacAfaauaagaG UCUCUUGCUCUCUU 5058 12232 22828 uacaL96 228283 faGfcaagasgsa AUUUGUACC 25.1 29.1 0.1 AD- A- 2359 csusugcuCfuCfUfUfauuugu A- 2808 VPusGfsguaCfaaauaagA CUCUUGCUCUCUUA 5059 12232 22828 accaL96 228283 fgAfgcaagsasg UUUGUACCG 26.1 31.1 2.1 AD- A- 2360 ususgcucUfcUfUfAfuuugua A- 2809 VPusCfsgguAfcaaauaaG UCUUGCUCUCUUAU 5060 12232 22828 ccgaL96 228283 faGfagcaasgsa UUGUACCGG 27.1 33.1 4.1 AD- A- 2361 usgscucuCfuUfAfUfuuguac A- 2810 VPusCfscggUfacaaauaA CUUGCUCUCUUAUU 5061 12232 22828 cggaL96 228283 fgAfgagcasasg UGUACCGGU 28.1 35.1 6.1 AD- A- 2362 csuscucuUfaUfUfUfguaccg A- 2811 VPusAfsaccGfguacaaaU UGCUCUCUUAUUUG 5062 12232 22828 guuaL96 228283 faAfgagagscsa UACCGGUUU 29.1 37.1 8.1 AD- A- 2363 uscsucuuAfuUfUfGfuaccgg A- 2812 VPusAfsaacCfgguacaaA GCUCUCUUAUUUGU 5063 12232 22828 uuuaL96 228284 fuAfagagasgsc ACCGGUUUU 30.1 39.1 0.1 AD- A- 2364 csuscuuaUfuUfGfUfaccggu A- 2813 VPusAfsaaaCfcgguacaA CUCUCUUAUUUGUA 5064 12232 22828 uuuaL96 228284 faUfaagagsasg CCGGUUUUU 31.1 41.1 2.1 AD- A- 2365 uscsuuauUfuGfUfAfccgguu A- 2814 VPusAfsaaaAfccgguacA UCUCUUAUUUGUAC 5065 12232 22828 uuuaL96 228284 faAfuaagasgsa CGGUUUUUG 32.1 43.1 4.1 AD- A- 2366 csusuauuUfgUfAfCfcgguuu A- 2815 VPusCfsaaaAfaccgguaC CUCUUAUUUGUACC 5066 12232 22828 uugaL96 228284 faAfauaagsasg GGUUUUUGU 33.1 45.1 6.1 AD- A- 2367 ususauuuGfuAfCfCfgguuu A- 2816 VPusAfscaaAfaaccgguA UCUUAUUUGUACCG 5067 12232 22828 uuguaL96 228284 fcAfaauaasgsa GUUUUUGUA 34.1 47.1 8.1 AD- A- 2368 usasuuugUfaCfCfGfguuuuu A- 2817 VPusUfsacaAfaaaccggU CUUAUUUGUACCGG 5068 12232 22828 guaaL96 228285 faCfaaauasasg UUUUUGUAU 35.1 49.1 0.1 AD- A- 2369 asusuuguAfcCfGfGfuuuuu A- 2818 VPusAfsuacAfaaaaccgG UUAUUUGUACCGGU 5069 12232 22828 guauaL96 228285 fuAfcaaausasa UUUUGUAUA 36.1 51.1 2.1 AD- A- 2370 ususuguaCfcGfGfUfuuuug A- 2819 VPusUfsauaCfaaaaaccG UAUUUGUACCGGUU 5070 12232 22828 uauaaL96 228285 fgUfacaaasusa UUUGUAUAU 37.1 53.1 4.1 AD- A- 2371 ususguacCfgGfUfUfuuugua A- 2820 VPusAfsuauAfcaaaaacC AUUUGUACCGGUUU 5071 12232 22828 uauaL96 228285 fgGfuacaasasu UUGUAUAUA 38.1 55.1 6.1 AD- A- 2372 usgsuaccGfgUfUfUfuuguau A- 2821 VPusUfsauaUfacaaaaaC UUUGUACCGGUUUU 5072 12232 22828 auaaL96 228285 fcGfguacasasa UGUAUAUAA 39.1 57.1 8.1 AD- A- 2373 gsusaccgGfuUfUfUfuguaua A- 2822 VPusUfsuauAfuacaaaaA UUGUACCGGUUUUU 5073 12232 22828 uaaaL96 228286 fcCfgguacsasa GUAUAUAAA 40.1 59.1 0.1 AD- A- 2374 usasccggUfuUfUfUfguauau A- 2823 VPusUfsuuaUfauacaaaA UGUACCGGUUUUUG 5074 12232 22828 aaaaL96 228286 faCfcgguascsa UAUAUAAAA 41.1 61.1 2.1 AD- A- 2375 ascscgguUfuUfUfGfuauaua A- 2824 VPusUfsuuuAfuauacaa GUACCGGUUUUUGU 5075 12232 22828 aaaaL96 228286 AfaAfccggusasc AUAUAAAAU 42.1 63.1 4.1 AD- A- 2376 asusucauGfuUfUfCfcaaucu A- 2825 VPusGfsagaGfauuggaa AAAUUCAUGUUUCC 5076 12232 22828 cucaL96 228286 AfcAfugaaususu AAUCUCUCU 43.1 65.1 6.1 AD- A- 2377 ususcaugUfuUfCfCfaaucuc A- 2826 VPusAfsgagAfgauugga AAUUCAUGUUUCCA 5077 12232 22828 ucuaL96 228286 AfaCfaugaasusu AUCUCUCUC 44.1 67.1 8.1 AD- A- 2378 uscsauguUfuCfCfAfaucucu A- 2827 VPusGfsagaGfagauugg AUUCAUGUUUCCAA 5078 12232 22828 cucaL96 228287 AfaAfcaugasasu UCUCUCUCU 45.1 69.1 0.1 AD- A- 2379 csasuguuUfcCfAfAfucucuc A- 2828 VPusAfsgagAfgagauug UUCAUGUUUCCAAU 5079 12232 22828 ucuaL96 228287 GfaAfacaugsasa CUCUCUCUC 46.1 71.1 2.1 AD- A- 2380 asusguuuCfcAfAfUfcucucu A- 2829 VPusGfsagaGfagagauu UCAUGUUUCCAAUC 5080 12232 22828 cucaL96 228287 GfgAfaacausgsa UCUCUCUCC 47.1 73.1 4.1 AD- A- 2381 usgsuuucCfaAfUfCfucucuc A- 2830 VPusGfsgagAfgagagau CAUGUUUCCAAUCU 5081 12232 22828 uccaL96 228287 UfgGfaaacasusg CUCUCUCCC 48.1 75.1 6.1 AD- A- 2382 ususuccaAfuCfUfCfucucuc A- 2831 VPusAfsgggAfgagagag UGUUUCCAAUCUCU 5082 12232 22828 ccuaL96 228287 AfuUfggaaascsa CUCUCCCUG 49.1 77.1 8.1 AD- A- 2383 uscscaauCfuCfUfCfucuccc A- 2832 VPusUfscagGfgagagag UUUCCAAUCUCUCU 5083 12232 22828 ugaaL96 228288 AfgAfuuggasasa CUCCCUGAU 50.1 79.1 0.1 AD- A- 2384 csgsgugaCfaGfUfCfacuagc A- 2833 VPusUfsaagCfuagugacU AUCGGUGACAGUCA 5084 12232 22828 uuaaL96 228288 fgUfcaccgsasu CUAGCUUAU 51.1 81.1 2.1 AD- A- 2385 gsgsugacAfgUfCfAfcuagcu A- 2834 VPusAfsuaaGfcuagugaC UCGGUGACAGUCAC 5085 12232 22828 uauaL96 228288 fuGfucaccsgsa UAGCUUAUC 52.1 83.1 4.1 AD- A- 2386 asgsucacUfaGfCfUfuaucuu A- 2835 VPusUfsucaAfgauaagcU ACAGUCACUAGCUU 5086 12232 22828 gaaaL96 228288 faGfugacusgsu AUCUUGAAC 53.1 85.1 6.1 AD- A- 2387 gsuscacuAfgCfUfUfaucuug A- 2836 VPusGfsuucAfagauaagC CAGUCACUAGCUUA 5087 12232 22828 aacaL96 228288 fuAfgugacsusg UCUUGAACA 54.1 87.1 8.1 AD- A- 2388 uscsacuaGfcUfUfAfucuuga A- 2837 VPusUfsguuCfaagauaaG AGUCACUAGCUUAU 5088 12232 22828 acaaL96 228289 fcUfagugascsu CUUGAACAG 55.1 89.1 0.1 AD- A- 2389 ascsuagcUfuAfUfCfuugaac A- 2838 VPusUfscugUfucaagau UCACUAGCUUAUCU 5089 12232 22828 agaaL96 228289 AfaGfcuagusgsa UGAACAGAU 56.1 91.1 2.1 AD- A- 2390 csusagcuUfaUfCfUfugaaca A- 2839 VPusAfsucuGfuucaaga CACUAGCUUAUCUU 5090 12232 22828 gauaL96 228289 UfaAfgcuagsusg GAACAGAUA 57.1 93.1 4.1 AD- A- 2391 usasgcuuAfuCfUfUfgaacag A- 2840 VPusUfsaucUfguucaag ACUAGCUUAUCUUG 5091 12232 22828 auaaL96 228289 AfuAfagcuasgsu AACAGAUAU 58.1 95.1 6.1 AD- A- 2392 asgscuuaUfcUfUfGfaacaga A- 2841 VPusAfsuauCfuguucaa CUAGCUUAUCUUGA 5092 12232 22828 uauaL96 228289 GfaUfaagcusasg ACAGAUAUU 59.1 97.1 8.1 AD- A- 2393 gscsuuauCfuUfGfAfacagau A- 2842 VPusAfsauaUfcuguuca UAGCUUAUCUUGAA 5093 12232 22828 auuaL96 228290 AfgAfuaagcsusa CAGAUAUUU 60.1 99.1 0.1 AD- A- 2394 csasgcacAfcAfUfUfccuuug A- 2843 VPusUfsuucAfaaggaau CCCAGCACACAUUC 5094 12232 22829 aaaaL96 228290 GfuGfugcugsgsg CUUUGAAAU 61.1 01.1 2.1 AD- A- 2395 asgscacaCfaUfUfCfcuuuga A- 2844 VPusAfsuuuCfaaaggaaU CCAGCACACAUUCC 5095 12232 22829 aauaL96 228290 fgUfgugcusgsg UUUGAAAUA 62.1 03.1 4.1 AD- A- 2396 gscsacacAfuUfCfCfuuugaa A- 2845 VPusUfsauuUfcaaaggaA CAGCACACAUUCCU 5096 12232 22829 auaaL96 228290 fuGfugugcsusg UUGAAAUAA 63.1 05.1 6.1 AD- A- 2397 csascacaUfuCfCfUfuugaaa A- 2846 VPusUfsuauUfucaaagg AGCACACAUUCCUU 5097 12232 22829 uaaaL96 228290 AfaUfgugugscsu UGAAAUAAG 64.1 07.1 8.1 AD- A- 2398 csascauuCfcUfUfUfgaaaua A- 2847 VPusCfscuuAfuuucaaaG CACACAUUCCUUUG 5098 12232 22829 aggaL96 228291 fgAfaugugsusg AAAUAAGGU 65.1 09.1 0.1 AD- A- 2399 uscscuuuGfaAfAfUfaagguu A- 2848 VPusUfsgaaAfccuuauu AUUCCUUUGAAAUA 5099 12232 22829 ucaaL96 228291 UfcAfaaggasasu AGGUUUCAA 66.1 11.1 2.1 AD- A- 2400 csusuugaAfaUfAfAfgguuuc A- 2849 VPusAfsuugAfaaccuua UCCUUUGAAAUAAG 5100 12232 22829 aauaL96 228291 UfuUfcaaagsgsa GUUUCAAUA 67.1 13.1 4.1 AD- A- 2401 ususugaaAfuAfAfGfguuuca A- 2850 VPusUfsauuGfaaaccuuA CCUUUGAAAUAAGG 5101 12232 22829 auaaL96 228291 fuUfucaaasgsg UUUCAAUAU 68.1 15.1 6.1 AD- A- 2402 asgsguuuCfaAfUfAfuacauc A- 2851 VPusGfsuagAfuguauau UAAGGUUUCAAUAU 5102 12232 22829 uacaL96 228291 UfgAfaaccususa ACAUCUACA 69.1 17.1 8.1 AD- A- 2403 gsgsuuucAfaUfAfUfacaucu A- 2852 VPusUfsguaGfauguaua AAGGUUUCAAUAUA 5103 12232 22829 acaaL96 228292 UfuGfaaaccsusu CAUCUACAU 70.1 19.1 0.1 AD- A- 2404 gsusuucaAfuAfUfAfcaucua A- 2853 VPusAfsuguAfgauguau AGGUUUCAAUAUAC 5104 12232 22829 cauaL96 228292 AfuUfgaaacscsu AUCUACAUA 71.1 21.1 2.1 AD- A- 2405 ususucaaUfaUfAfCfaucuac A- 2854 VPusUfsaugUfagaugua GGUUUCAAUAUACA 5105 12232 22829 auaaL96 228292 UfaUfugaaascsc UCUACAUAC 72.1 23.1 4.1 AD- A- 2406 ususcaauAfuAfCfAfucuaca A- 2855 VPusGfsuauGfuagaugu GUUUCAAUAUACAU 5106 12232 22829 uacaL96 228292 AfuAfuugaasasc CUACAUACU 73.1 25.1 6.1 AD- A- 2407 usasuuugGfcAfAfCfuuguau A- 2856 VPusCfsaaaUfacaaguuG UAUAUUUGGCAACU 5107 12232 22829 uugaL96 228292 fcCfaaauasusa UGUAUUUGU 74.1 27.1 8.1 AD- A- 2408 ususuggcAfaCfUfUfguauuu A- 2857 VPusCfsacaAfauacaagU UAUUUGGCAACUUG 5108 12232 22829 gugaL96 228293 fuGfccaaasusa UAUUUGUGU 75.1 29.1 0.1 AD- A- 2409 usgsgcaaCfuUfGfUfauuugu A- 2858 VPusCfsacaCfaaauacaA UUUGGCAACUUGUA 5109 12232 22829 gugaL96 228293 fgUfugccasasa UUUGUGUGU 76.1 31.1 2.1 AD- A- 2410 gsgscaacUfuGfUfAfuuugug A- 2859 VPusAfscacAfcaaauacA UUGGCAACUUGUAU 5110 12232 22829 uguaL96 228293 faGfuugccsasa UUGUGUGUA 77.1 33.1 4.1 AD- A- 2411 gscsaacuUfgUfAfUfuugugu A- 2860 VPusUfsacaCfacaaauaC UGGCAACUUGUAUU 5111 12232 22829 guaaL96 228293 faAfguugcscsa UGUGUGUAU 78.1 35.1 6.1 AD- A- 2412 csasacuuGfuAfUfUfugugug A- 2861 VPusAfsuacAfcacaaauA GGCAACUUGUAUUU 5112 12232 22829 uauaL96 228293 fcAfaguugscsc GUGUGUAUA 79.1 37.1 8.1 AD- A- 2413 asascuugUfaUfUfUfgugugu A- 2862 VPusUfsauaCfacacaaaU GCAACUUGUAUUUG 5113 12232 22829 auaaL96 228294 faCfaaguusgsc UGUGUAUAU 80.1 39.1 0.1 AD- A- 2414 ascsuuguAfuUfUfGfugugu A- 2863 VPusAfsuauAfcacacaaA CAACUUGUAUUUGU 5114 12232 22829 auauaL96 228294 fuAfcaagususg GUGUAUAUA 81.1 41.1 2.1 AD- A- 2415 ususcugaUfaAfAfAfuagaca A- 2864 VPusCfsaauGfucuauuu GAUUCUGAUAAAAU 5115 12232 22829 uugaL96 228294 UfaUfcagaasusc AGACAUUGC 82.1 43.1 4.1 AD- A- 2416 usgsauaaAfaUfAfGfacauug A- 2865 VPusUfsagcAfaugucua UCUGAUAAAAUAGA 5116 12232 22829 cuaaL96 228294 UfuUfuaucasgsa CAUUGCUAU 83.1 45.1 6.1 AD- A- 2417 gsasuaaaAfuAfGfAfcauugc A- 2866 VPusAfsuagCfaaugucu CUGAUAAAAUAGAC 5117 12232 22829 uauaL96 228294 AfuUfuuaucsasg AUUGCUAUU 84.1 47.1 8.1 AD- A- 2418 usasaaauAfgAfCfAfuugcua A- 2867 VPusGfsaauAfgcaauguC GAUAAAAUAGACAU 5118 12232 22829 uucaL96 228295 fuAfuuuuasusc UGCUAUUCU 85.1 49.1 0.1 AD- A- 2419 usasgacaUfuGfCfUfauucug A- 2868 VPusAfsaacAfgaauagcA AAUAGACAUUGCUA 5119 12232 22829 uuuaL96 228295 faUfgucuasusu UUCUGUUUU 86.1 51.1 2.1 AD- A- 2420 asgsacauUfgCfUfAfuucugu A- 2869 VPusAfsaaaCfagaauagC AUAGACAUUGCUAU 5120 12232 22829 uuuaL96 228295 faAfugucusasu UCUGUUUUU 87.1 53.1 4.1 AD- A- 2421 uscsuacaUfaCfUfAfaaucuc A- 2870 VPusAfsgagAfgauuuag AUUCUACAUACUAA 5121 12232 22829 ucuaL96 228295 UfaUfguagasasu AUCUCUCUC 88.1 55.1 6.1 AD- A- 2422 csusacauAfcUfAfAfaucucu A- 2871 VPusGfsagaGfagauuua UUCUACAUACUAAA 5122 12232 22829 cucaL96 228295 GfuAfuguagsasa UCUCUCUCC 89.1 57.1 8.1 AD- A- 2423 usascauaCfuAfAfAfucucuc A- 2872 VPusGfsgagAfgagauuu UCUACAUACUAAAU 5123 12232 22829 uccaL96 228296 AfgUfauguasgsa cucucuccu 90.1 59.1 0.1 AD- A- 2424 ascsauacUfaAfAfUfcucucu A- 2873 VPusAfsggaGfagagauu CUACAUACUAAAUC 5124 12232 22829 ccuaL96 228296 UfaGfuaugusasg UCUCUCCUU 91.1 61.1 2.1 AD- A- 2425 csasuacuAfaAfUfCfucucuc A- 2874 VPusAfsaggAfgagagau UACAUACUAAAUCU 5125 12232 22829 cuuaL96 228296 UfuAfguaugsusa CUCUCCUUU 92.1 63.1 4.1 AD- A- 2426 asusacuaAfaUfCfUfcucucc A- 2875 VPusAfsaagGfagagagaU ACAUACUAAAUCUC 5126 12232 22829 uuuaL96 228296 fuUfaguausgsu UCUCCUUUU 93.1 65.1 6.1 AD- A- 2427 usascuaaAfuCfUfCfucuccu A- 2876 VPusAfsaaaGfgagagagA CAUACUAAAUCUCU 5127 12232 22829 uuuaL96 228296 fuUfuaguasusg CUCCUUUUU 94.1 67.1 8.1 AD- A- 2428 csasuuuaUfuUfAfUfuggugc A- 2877 VPusGfsuagCfaccaauaA AUCAUUUAUUUAUU 5128 12232 22829 uacaL96 228297 faUfaaaugsasu GGUGCUACU 95.1 69.1 0.1 AD- A- 2429 asusuuauUfuAfUfUfggugc A- 2878 VPusAfsguaGfcaccaauA UCAUUUAUUUAUUG 5129 12232 22829 uacuaL96 228297 faAfuaaausgsa GUGCUACUG 96.1 71.1 2.1 AD- A- 2430 ususuauuUfaUfUfGfgugcua A- 2879 VPusCfsaguAfgcaccaaU CAUUUAUUUAUUGG 5130 12232 22829 cugaL96 228297 faAfauaaasusg UGCUACUGU 97.1 73.1 4.1 AD- A- 2431 ususauuuAfuUfGfGfugcuac A- 2880 VPusAfscagUfagcaccaA AUUUAUUUAUUGG 5131 12232 22829 uguaL96 228297 fuAfaauaasasu UGCUACUGUU 98.1 75.1 6.1 AD- A- 2432 usasuuuaUfuGfGfUfgcuacu A- 2881 VPusAfsacaGfuagcaccA UUUAUUUAUUGGU 5132 12232 22829 guuaL96 228297 faUfaaauasasa GCUACUGUUU 99.1 77.1 8.1 AD- A- 2433 asusuuauUfgGfUfGfcuacug A- 2882 VPusAfsaacAfguagcacC UUAUUUAUUGGUGC 5133 12233 22829 uuuaL96 228298 faAfuaaausasa UACUGUUUA 00.1 79.1 0.1 AD- A- 2434 ususauugGfuGfCfUfacuguu A- 2883 VPusAfsuaaAfcaguagcA AUUUAUUGGUGCUA 5134 12233 22829 uauaL96 228298 fcCfaauaasasu CUGUUUAUC 01.1 81.1 2.1 AD- A- 2435 asusugguGfcUfAfCfuguuua A- 2884 VPusGfsgauAfaacaguaG UUAUUGGUGCUACU 5135 12233 22829 uccaL96 228298 fcAfccaausasa GUUUAUCCG 02.1 83.1 4.1 AD- A- 2436 gsasaaagAfuAfUfUfaacauc A- 2885 VPusCfsgugAfuguuaau GGGAAAAGAUAUU 5136 12233 22829 acgaL96 228298 AfuCfuuuucscsc AACAUCACGU 03.1 85.1 6.1 AD- A- 2437 asascaucAfcGfUfCfuuuguc A- 2886 VPusAfsgagAfcaaagacG UUAACAUCACGUCU 5137 12233 22829 ucuaL96 228298 fuGfauguusasa UUGUCUCUA 04.1 87.1 8.1 AD- A- 2438 ascsaucaCfgUfCfUfuugucu A- 2887 VPusUfsagaGfacaaagaC UAACAUCACGUCUU 5138 12233 22829 cuaaL96 228299 fgUfgaugususa UGUCUCUAG 05.1 89.1 0.1 AD- A- 2439 gsuscuuuGfuCfUfCfuagugc A- 2888 VPusAfscugCfacuagagA ACGUCUUUGUCUCU 5139 12233 22829 aguaL96 228299 fcAfaagacsgsu AGUGCAGUU 06.1 91.1 2.1 AD- A- 2440 uscsuuugUfcUfCfUfagugca A- 2889 VPusAfsacuGfcacuagaG CGUCUUUGUCUCUA 5140 12233 22829 guuaL96 228299 faCfaaagascsg GUGCAGUUU 07.1 93.1 4.1 AD- A- 2441 csusuuguCfuCfUfAfgugcag A- 2890 VPusAfsaacUfgcacuagA GUCUUUGUCUCUAG 5141 12233 22829 uuuaL96 228299 fgAfcaaagsasc UGCAGUUUU 08.1 95.1 6.1 AD- A- 2442 gsasgauaUfuCfCfGfuaguac A- 2891 VPusUfsaugUfacuacgg UCGAGAUAUUCCGU 5142 12233 22829 auaaL96 228299 AfaUfaucucsgsa AGUACAUAU 09.1 97.1 8.1 AD- A- 2443 asgsauauUfcCfGfUfaguaca A- 2892 VPusAfsuauGfuacuacg CGAGAUAUUCCGUA 5143 12233 22829 uauaL96 228300 GfaAfuaucuscsg GUACAUAUU 10.1 99.1 0.1 AD- A- 2444 gsasuauuCfcGfUfAfguacau A- 2893 VPusAfsauaUfguacuacG GAGAUAUUCCGUAG 5144 12233 22830 auuaL96 228300 fgAfauaucsusc UACAUAUUU 11.1 01.1 2.1 AD- A- 2445 csgsacaaAfgAfAfAfuacaga A- 2894 VPusAfsuauCfuguauuu AACGACAAAGAAAU 5145 12233 22830 uauaL96 228300 CfuUfugucgsusu ACAGAUAUA 12.1 03.1 4.1 AD- A- 2446 gsascaaaGfaAfAfUfacagau A- 2895 VPusUfsauaUfcuguauu ACGACAAAGAAAUA 5146 12233 22830 auaaL96 228300 UfcUfuugucsgsu CAGAUAUAU 13.1 05.1 6.1 AD- A- 2447 ascsaaagAfaAfUfAfcagaua A- 2896 VPusAfsuauAfucuguau CGACAAAGAAAUAC 5147 12233 22830 uauaL96 228300 UfuCfuuuguscsg AGAUAUAUC 14.1 07.1 8.1 AD- A- 2448 csasaagaAfaUfAfCfagauau A- 2897 VPusGfsauaUfaucugua GACAAAGAAAUACA 5148 12233 22830 aucaL96 228301 UfuUfcuuugsusc GAUAUAUCU 15.1 09.1 0.1

TABLE 8B Exemplary Human VEGF-A siRNA Unmodified Single Strands and Duplex Sequences Anti- SEQ ID Sense SEQ ID mRNA sense NO: mRNA Duplex Oligo NO: Target Oligo (Anti- Target Name Name (Sense) Sense Sequence Range Name sense) Antisense Sequence Range AD- A- 2898 CGGUGCUGGAAUUU  123- A- 3347 UAAUAUCAAAUUCCA  121- 122286 228211 GAUAUUA  143 2282112. GCACCGAG  143 6.1 1.1 1 AD- A- 2899 GGUGCUGGAAUUUG  124- A- 3348 UGAAUAUCAAAUUCC  122- 122286 228211 AUAUUCA  144 2282114. AGCACCGA  144 7.1 3.1 1 AD- A- 2900 UGCUGGAAUUUGAU  126- A- 3349 UAUGAAUAUCAAAUU  124- 122286 228211 AUUCAUA  146 2282116. CCAGCACC  146 8.1 5.1 1 AD- A- 2901 GCUGGAAUUUGAUA  127- A- 3350 UAAUGAAUAUCAAAU  125- 122286 228211 UUCAUUA  147 2282118. UCCAGCAC  147 9.1 7.1 1 AD- A- 2902 UGGAAUUUGAUAUU  129- A- 3351 UUCAAUGAAUAUCAA  127- 122287 228211 CAUUGAA  149 2282120. AUUCCAGC  149 0.1 9.1 1 AD- A- 2903 GGAAUUUGAUAUUC  130- A- 3352 UAUCAAUGAAUAUCA  128- 122287 228212 AUUGAUA  150 2282122. AAUUCCAG  150 1.1 1.1 1 AD- A- 2904 GAAUUUGAUAUUCA  131- A- 3353 UGAUCAAUGAAUAUC  129- 122287 228212 UUGAUCA  151 2282124. AAAUUCCA  151 2.1 3.1 1 AD- A- 2905 AAUUUGAUAUUCAU  132- A- 3354 UGGAUCAAUGAAUAU  130- 122287 228212 UGAUCCA  152 2282126. CAAAUUCC  152 3.1 5.1 1 AD- A- 2906 AUUUGAUAUUCAUU  133- A- 3355 UCGGAUCAAUGAAUA  131- 122287 228212 GAUCCGA  153 2282128. UCAAAUUC  153 4.1 7.1 1 AD- A- 2907 UUUGAUAUUCAUUG  134- A- 3356 UCCGGAUCAAUGAAU  132- 122287 228212 AUCCGGA  154 2282130. AUCAAAUU  154 5.1 9.1 1 AD- A- 2908 UUUAUUUUUGCUUG  217- A- 3357 UGAAUGGCAAGCAAA  215- 122287 228213 CCAUUCA  237 2282132. AAUAAAUU  237 6.1 1.1 1 AD- A- 2909 UUAUUUUUGCUUGCC  218- A- 3358 UGGAAUGGCAAGCAA  216- 122287 228213 AUUCCA  238 2282134. AAAUAAAU  238 7.1 3.1 1 AD- A- 2910 CAAAUCACUGUGGAU  283- A- 3359 UCCAAAAUCCACAGU  281- 122287 228213 UUUGGA  303 2282136. GAUUUGGG  303 8.1 5.1 1 AD- A- 2911 AAAUCACUGUGGAU  284- A- 3360 UUCCAAAAUCCACAG  282- 122287 228213 UUUGGAA  304 2282138. UGAUUUGG  304 9.1 7.1 1 AD- A- 2912 AAUCACUGUGGAUU  285- A- 3361 UUUCCAAAAUCCACA  283- 122288 228213 UUGGAAA  305 2282140. GUGAUUUG  305 0.1 9.1 1 AD- A- 2913 AUCACUGUGGAUUU  286- A- 3362 UUUUCCAAAAUCCAC  284- 122288 228214 UGGAAAA  306 2282142. AGUGAUUU  306 1.1 1.1 1 AD- A- 2914 UCACUGUGGAUUUU  287- A- 3363 UGUUUCCAAAAUCCA  285- 122288 228214 GGAAACA  307 2282144. CAGUGAUU  307 2.1 3.1 1 AD- A- 2915 CACUGUGGAUUUUG  288- A- 3364 UGGUUUCCAAAAUCC  286- 122288 228214 GAAACCA  308 2282146. ACAGUGAU  308 3.1 5.1 1 AD- A- 2916 ACUGUGGAUUUUGG  289- A- 3365 UUGGUUUCCAAAAUC  287- 122288 228214 AAACCAA  309 2282148. CACAGUGA  309 4.1 7.1 1 AD- A- 2917 CUGUGGAUUUUGGA  290- A- 3366 UCUGGUUUCCAAAAU  288- 122288 228214 AACCAGA  310 2282150. CCACAGUG  310 5.1 9.1 1 AD- A- 2918 UGUGGAUUUUGGAA  291- A- 3367 UGCUGGUUUCCAAAA  289- 122288 228215 ACCAGCA  311 2282152. UCCACAGU  311 6.1 1.1 1 AD- A- 2919 GUGGAUUUUGGAAA  292- A- 3368 UUGCUGGUUUCCAAA  290- 122288 228215 CCAGCAA  312 2282154. AUCCACAG  312 7.1 3.1 1 AD- A- 2920 GAUUUUGGAAACCA  295- A- 3369 UUUCUGCUGGUUUCC  293- 122288 228215 GCAGAAA  315 2282156. AAAAUCCA  315 8.1 5.1 1 AD- A- 2921 AUUUUGGAAACCAGC  296- A- 3370 UUUUCUGCUGGUUUC  294- 122288 228215 AGAAAA  316 2282158. CAAAAUCC  316 9.1 7.1 1 AD- A- 2922 UUUUGGAAACCAGCA  297- A- 3371 UCUUUCUGCUGGUUU  295- 122289 228215 GAAAGA  317 2282160. CCAAAAUC  317 0.1 9.1 1 AD- A- 2923 UUUGGAAACCAGCAG  298- A- 3372 UUCUUUCUGCUGGUU  296- 122289 228216 AAAGAA  318 2282162. UCCAAAAU  318 1.1 1.1 1 AD- A- 2924 GGAAACCAGCAGAAA  301- A- 3373 UUCCUCUUUCUGCUG  299- 122289 228216 GAGGAA  321 2282164. GUUUCCAA  321 2.1 3.1 1 AD- A- 2925 AAACCAGCAGAAAGA  303- A- 3374 UUUUCCUCUUUCUGC  301- 122289 228216 GGAAAA  323 2282166. UGGUUUCC  323 3.1 5.1 1 AD- A- 2926 AACCAGCAGAAAGAG  304- A- 3375 UCUUUCCUCUUUCUG  302- 122289 228216 GAAAGA  324 2282168. CUGGUUUC  324 4.1 7.1 1 AD- A- 2927 ACCAGCAGAAAGAGG  305- A- 3376 UUCUUUCCUCUUUCU  303- 122289 228216 AAAGAA  325 2282170. GCUGGUUU  325 5.1 9.1 1 AD- A- 2928 CCAGCAGAAAGAGGA  306- A- 3377 UCUCUUUCCUCUUUC  304- 122289 228217 AAGAGA  326 2282172. UGCUGGUU  326 6.1 1.1 1 AD- A- 2929 CAGCAGAAAGAGGA  307- A- 3378 uccucuuuccucuuuc  305- 122289 228217 AAGAGGA  327 2282174. UGCUGGU  327 7.1 3.1 1 AD- A- 2930 AGCAGAAAGAGGAA  308- A- 3379 UACCUCUUUCCUCUU  306- 122289 228217 AGAGGUA  328 2282176. UCUGCUGG  328 8.1 5.1 1 AD- A- 2931 GCAGAAAGAGGAAA  309- A- 3380 UUACCUCUUUCCUCU  307- 122289 228217 GAGGUAA  329 2282178. UUCUGCUG  329 9.1 7.1 1 AD- A- 2932 CAGAAAGAGGAAAG  310- A- 3381 UCUACCUCUUUCCUCU  308- 122290 228217 AGGUAGA  330 2282180. UUCUGCU  330 0.1 9.1 1 AD- A- 2933 AAAGAGGAAAGAGG  313- A- 3382 UUUGCUACCUCUUUC  311- 122290 228218 UAGCAAA  333 2282182. CUCUUUCU  333 1.1 1.1 1 AD- A- 2934 AAGAGGAAAGAGGU  314- A- 3383 UCUUGCUACCUCUUU  312- 122290 228218 AGCAAGA  334 2282184. ccucuuuc  334 2.1 3.1 1 AD- A- 2935 AGAGGAAAGAGGUA  315- A- 3384 UUCUUGCUACCUCUU  313- 122290 228218 GCAAGAA  335 2282186. uccucuuu  335 3.1 5.1 1 AD- A- 2936 GAGGAAAGAGGUAG  316- A- 3385 UCUCUUGCUACCUCU  314- 122290 228218 CAAGAGA  336 2282188. uuccucuu  336 4.1 7.1 1 AD- A- 2937 GGAAAGAGGUAGCA  318- A- 3386 UAGCUCUUGCUACCU  316- 122290 228218 AGAGCUA  338 2282190. cuuuccuc  338 5.1 9.1 1 AD- A- 2938 AGGUAGCAAGAGCUC  324- A- 3387 UCUCUGGAGCUCUUG  322- 122290 228219 CAGAGA  344 2282192. CUACCUCU  344 6.1 1.1 1 AD- A- 2939 UCCAGAGAGAAGUCG  337- A- 3388 UUUCCUCGACUUCUC  335- 122290 228219 AGGAAA  357 2282194. UCUGGAGC  357 7.1 3.1 1 AD- A- 2940 CCAGAGAGAAGUCGA  338- A- 3389 UCUUCCUCGACUUCUC  336- 122290 228219 GGAAGA  358 2282196. UCUGGAG  358 8.1 5.1 1 AD- A- 2941 CAGAGAGAAGUCGA  339- A- 3390 UUCUUCCUCGACUUC  337- 122290 228219 GGAAGAA  359 2282198. UCUCUGGA  359 9.1 7.1 1 AD- A- 2942 AGAGAAGUCGAGGA  342- A- 3391 UCUCUCUUCCUCGACU  340- 122291 228219 AGAGAGA  362 2282200. UCUCUCU  362 0.1 9.1 1 AD- A- 2943 GAGAAGUCGAGGAA  343- A- 3392 UUCUCUCUUCCUCGAC  341- 122291 228220 GAGAGAA  363 2282202. UUCUCUC  363 1.1 1.1 1 AD- A- 2944 GAAGUCGAGGAAGA  345- A- 3393 UUCUCUCUCUUCCUCG  343- 122291 228220 GAGAGAA  365 2282204. ACUUCUC  365 2.1 3.1 1 AD- A- 2945 AGUGAGUGACCUGCU  417- A- 3394 UCCAAAAGCAGGUCA  415- 122291 228220 UUUGGA  437 2282206. CUCACUUU  437 3.1 5.1 1 AD- A- 2946 GGCGUCGCACUGAAA  643- A- 3395 UAAAAGUUUCAGUGC  641- 122291 228220 CUUUUA  663 2282208. GACGCCGC  663 4.1 7.1 1 AD- A- 2947 GUCGCACUGAAACUU  646- A- 3396 UACGAAAAGUUUCAG  644- 122291 228220 UUCGUA  666 2282210. UGCGACGC  666 5.1 9.1 1 AD- A- 2948 UCGCACUGAAACUUU  647- A- 3397 UGACGAAAAGUUUCA  645- 122291 228221 UCGUCA  667 2282212. GUGCGACG  667 6.1 1.1 1 AD- A- 2949 CACUGAAACUUUUCG  650- A- 3398 UUUGGACGAAAAGUU  648- 122291 228221 UCCAAA  670 2282214. UCAGUGCG  670 7.1 3.1 1 AD- A- 2950 ACUGAAACUUUUCGU  651- A- 3399 UGUUGGACGAAAAGU  649- 122291 228221 CCAACA  671 2282216. UUCAGUGC  671 8.1 5.1 1 AD- A- 2951 UGAAACUUUUCGUCC  653- A- 3400 UAAGUUGGACGAAAA  651- 122292 228221 AACUUA  673 2282220. GUUUCAGU  673 0.1 9.1 1 AD- A- 2952 AACUUUUCGUCCAAC  656- A- 3401 UCAGAAGUUGGACGA  654- 122292 228222 UUCUGA  676 2282222. AAAGUUUC  676 1.1 1.1 1 AD- A- 2953 CUGGGCUGUUCUCGC  673- A- 3402 UCCGAAGCGAGAACA  671- 122292 228222 UUCGGA  693 2282224. GCCCAGAA  693 2.1 3.1 1 AD- A- 2954 UGGGCUGUUCUCGCU  674- A- 3403 UUCCGAAGCGAGAAC  672- 122292 228222 UCGGAA  694 2282226. AGCCCAGA  694 3.1 5.1 1 AD- A- 2955 GGGCUGUUCUCGCUU  675- A- 3404 UCUCCGAAGCGAGAA  673- 122292 228222 CGGAGA  695 2282228. CAGCCCAG  695 4.1 7.1 1 AD- A- 2956 GCUGUUCUCGCUUCG  677- A- 3405 UUCCUCCGAAGCGAG  675- 122292 228222 GAGGAA  697 2282230. AACAGCCC  697 5.1 9.1 1 AD- A- 2957 GCCGCGAGAAGUGCU  733- A- 3406 UGAGCUAGCACUUCU  731- 122292 228223 AGCUCA  753 2282232. CGCGGCUC  753 6.1 1.1 1 AD- A- 2958 CCGCGAGAAGUGCUA  734- A- 3407 UCGAGCUAGCACUUC  732- 122292 228223 GCUCGA  754 2282234. UCGCGGCU  754 7.1 3.1 1 AD- A- 2959 GCCUCCGAAACCAUG 1027- A- 3408 UAAGUUCAUGGUUUC 1025- 122292 228223 AACUUA 1047 2282236. GGAGGCCC 1047 8.1 5.1 1 AD- A- 2960 AAGGAGGAGGGCAG 1130- A- 3409 UAUGAUUCUGCCCUC 1128- 122292 228223 AAUCAUA 1150 2282238. CUCCUUCU 1150 9.1 7.1 1 AD- A- 2961 GGCAGAAUCAUCACG 1139- A- 3410 UCACUUCGUGAUGAU 1137- 122293 228223 AAGUGA 1159 2282240. UCUGCCCU 1159 0.1 9.1 1 AD- A- 2962 AAUCAUCACGAAGUG 1144- A- 3411 UUUCACCACUUCGUG 1142- 122293 228224 GUGAAA 1164 2282242. AUGAUUCU 1164 1.1 1.1 1 AD- A- 2963 AUCAUCACGAAGUGG 1145- A- 3412 UCUUCACCACUUCGU 1143- 122293 228224 UGAAGA 1165 2282244. GAUGAUUC 1165 2.1 3.1 1 AD- A- 2964 UCAUCACGAAGUGGU 1146- A- 3413 UACUUCACCACUUCG 1144- 122293 228224 GAAGUA 1166 2282246. UGAUGAUU 1166 3.1 5.1 1 AD- A- 2965 CAUCACGAAGUGGUG 1147- A- 3414 UAACUUCACCACUUC 1145- 122293 228224 AAGUUA 1167 2282248. GUGAUGAU 1167 4.1 7.1 1 AD- A- 2966 UCACGAAGUGGUGA 1149- A- 3415 UUGAACUUCACCACU 1147- 122293 228224 AGUUCAA 1169 2282250. UCGUGAUG 1169 5.1 9.1 1 AD- A- 2967 AAGUGGUGAAGUUC 1154- A- 3416 UAUCCAUGAACUUCA 1152- 122293 228225 AUGGAUA 1174 2282252. CCACUUCG 1174 6.1 1.1 1 AD- A- 2968 AGUGGUGAAGUUCA 1155- A- 3417 UCAUCCAUGAACUUC 1153- 122293 228225 UGGAUGA 1175 2282254. ACCACUUC 1175 7.1 3.1 1 AD- A- 2969 GUGGUGAAGUUCAU 1156- A- 3418 UACAUCCAUGAACUU 1154- 122293 228225 GGAUGUA 1176 2282256. CACCACUU 1176 8.1 5.1 1 AD- A- 2970 GGUGAAGUUCAUGG 1158- A- 3419 UAGACAUCCAUGAAC 1156- 122293 228225 AUGUCUA 1178 2282258. UUCACCAC 1178 9.1 7.1 1 AD- A- 2971 GUGAAGUUCAUGGA 1159- A- 3420 UUAGACAUCCAUGAA 1157- 122294 228225 UGUCUAA 1179 2282260. CUUCACCA 1179 0.1 9.1 1 AD- A- 2972 UGAAGUUCAUGGAU 1160- A- 3421 UAUAGACAUCCAUGA 1158- 122294 228226 GUCUAUA 1180 2282262. ACUUCACC 1180 1.1 1.1 1 AD- A- 2973 AAGUUCAUGGAUGU 1162- A- 3422 UUGAUAGACAUCCAU 1160- 122294 228226 CUAUCAA 1182 2282264. GAACUUCA 1182 2.1 3.1 1 AD- A- 2974 AGUUCAUGGAUGUC 1163- A- 3423 UCUGAUAGACAUCCA 1161- 122294 228226 UAUCAGA 1183 2282266. UGAACUUC 1183 3.1 5.1 1 AD- A- 2975 UUCAUGGAUGUCUA 1165- A- 3424 UCGCUGAUAGACAUC 1163- 122294 228226 UCAGCGA 1185 2282268. CAUGAACU 1185 4.1 7.1 1 AD- A- 2976 GUGGACAUCUUCCAG 1213- A- 3425 UUACUCCUGGAAGAU 1211- 122294 228226 GAGUAA 1233 2282270. GUCCACCA 1233 5.1 9.1 1 AD- A- 2977 UCGAGUACAUCUUCA 1244- A- 3426 UUGGCUUGAAGAUGU 1242- 122294 228227 AGCCAA 1264 2282272. ACUCGAUC 1264 6.1 1.1 1 AD- A- 2978 CGAGUACAUCUUCAA 1245- A- 3427 UAUGGCUUGAAGAUG 1243- 122294 228227 GCCAUA 1265 2282274. UACUCGAU 1265 7.1 3.1 1 AD- A- 2979 GAGUACAUCUUCAAG 1246- A- 3428 UGAUGGCUUGAAGAU 1244- 122294 228227 CCAUCA 1266 2282276. GUACUCGA 1266 8.1 5.1 1 AD- A- 2980 GUACAUCUUCAAGCC 1248- A- 3429 UAGGAUGGCUUGAAG 1246- 122294 228227 AUCCUA 1268 2282278. AUGUACUC 1268 9.1 7.1 1 AD- A- 2981 CCAACAUCACCAUGC 1337- A- 3430 UAAUCUGCAUGGUGA 1335- 122295 228227 AGAUUA 1357 2282280. UGUUGGAC 1357 0.1 9.1 1 AD- A- 2982 CAACAUCACCAUGCA 1338- A- 3431 UUAAUCUGCAUGGUG 1336- 122295 228228 GAUUAA 1358 2282282. AUGUUGGA 1358 1.1 1.1 1 AD- A- 2983 ACAUCACCAUGCAGA 1340- A- 3432 UCAUAAUCUGCAUGG 1338- 122295 228228 UUAUGA 1360 2282284. UGAUGUUG 1360 2.1 3.1 1 AD- A- 2984 CAUCACCAUGCAGAU 1341- A- 3433 UGCAUAAUCUGCAUG 1339- 122295 228228 UAUGCA 1361 2282286. GUGAUGUU 1361 3.1 5.1 1 AD- A- 2985 ACCAUGCAGAUUAUG 1345- A- 3434 UAUCCGCAUAAUCUG 1343- 122295 228228 CGGAUA 1365 2282288. CAUGGUGA 1365 4.1 7.1 1 AD- A- 2986 AUGCAGAUUAUGCG 1348- A- 3435 UUUGAUCCGCAUAAU 1346- 122295 228228 GAUCAAA 1368 2282290. CUGCAUGG 1368 5.1 9.1 1 AD- A- 2987 UGCAGAUUAUGCGG 1349- A- 3436 UUUUGAUCCGCAUAA 1347- 122295 228229 AUCAAAA 1369 2282292. UCUGCAUG 1369 6.1 1.1 1 AD- A- 2988 GCAGAUUAUGCGGA 1350- A- 3437 UGUUUGAUCCGCAUA 1348- 122295 228229 UCAAACA 1370 2282294. AUCUGCAU 1370 7.1 3.1 1 AD- A- 2989 GAUUAUGCGGAUCA 1353- A- 3438 UGAGGUUUGAUCCGC 1351- 122295 228229 AACCUCA 1373 2282296. AUAAUCUG 1373 8.1 5.1 1 AD- A- 2990 AUUAUGCGGAUCAA 1354- A- 3439 UUGAGGUUUGAUCCG 1352- 122295 228229 ACCUCAA 1374 2282298. CAUAAUCU 1374 9.1 7.1 1 AD- A- 2991 CGGAUCAAACCUCAC 1360- A- 3440 UCCUUGGUGAGGUUU 1358- 122296 228229 CAAGGA 1380 2282300. GAUCCGCA 1380 0.1 9.1 1 AD- A- 2992 GGAGAGAUGAGCUU 1390- A- 3441 UUGUAGGAAGCUCAU 1388- 122296 228230 CCUACAA 1410 2282302. CUCUCCUA 1410 1.1 1.1 1 AD- A- 2993 GAGAUGAGCUUCCUA 1393- A- 3442 UUGCUGUAGGAAGCU 1391- 122296 228230 CAGCAA 1413 2282304. CAUCUCUC 1413 2.1 3.1 1 AD- A- 2994 GAUGAGCUUCCUACA 1395- A- 3443 UUGUGCUGUAGGAAG 1393- 122296 228230 GCACAA 1415 2282306. CUCAUCUC 1415 3.1 5.1 1 AD- A- 2995 AUGAGCUUCCUACAG 1396- A- 3444 UUUGUGCUGUAGGAA 1394- 122296 228230 CACAAA 1416 2282308. GCUCAUCU 1416 4.1 7.1 1 AD- A- 2996 GAGCUUCCUACAGCA 1398- A- 3445 UUGUUGUGCUGUAGG 1396- 122296 228230 CAACAA 1418 2282310. AAGCUCAU 1418 5.1 9.1 1 AD- A- 2997 AGCUUCCUACAGCAC 1399- A- 3446 UUUGUUGUGCUGUAG 1397- 122296 228231 AACAAA 1419 2282312. GAAGCUCA 1419 6.1 1.1 1 AD- A- 2998 GCUUCCUACAGCACA 1400- A- 3447 UUUUGUUGUGCUGUA 1398- 122296 228231 ACAAAA 1420 2282314. GGAAGCUC 1420 7.1 3.1 1 AD- A- 2999 CUUCCUACAGCACAA 1401- A- 3448 UAUUUGUUGUGCUGU 1399- 122296 228231 CAAAUA 1421 2282316. AGGAAGCU 1421 8.1 5.1 1 AD- A- 3000 UCCUACAGCACAACA 1403- A- 3449 UACAUUUGUUGUGCU 1401- 122296 228231 AAUGUA 1423 2282318. GUAGGAAG 1423 9.1 7.1 1 AD- A- 3001 CUACAGCACAACAAA 1405- A- 3450 UUCACAUUUGUUGUG 1403- 122297 228231 UGUGAA 1425 2282320. CUGUAGGA 1425 0.1 9.1 1 AD- A- 3002 UACAGCACAACAAAU 1406- A- 3451 UUUCACAUUUGUUGU 1404- 122297 228232 GUGAAA 1426 2282322. GCUGUAGG 1426 1.1 1.1 1 AD- A- 3003 AGCACAACAAAUGUG 1409- A- 3452 UGCAUUCACAUUUGU 1407- 122297 228232 AAUGCA 1429 2282324. UGUGCUGU 1429 2.1 3.1 1 AD- A- 3004 GCACAACAAAUGUGA 1410- A- 3453 UUGCAUUCACAUUUG 1408- 122297 228232 AUGCAA 1430 2282326. UUGUGCUG 1430 3.1 5.1 1 AD- A- 3005 CUCACCAGGAAAGAC 1786- A- 3454 UUAUCAGUCUUUCCU 1784- 122297 228232 UGAUAA 1806 2282328. GGUGAGAG 1806 4.1 7.1 1 AD- A- 3006 UCACCAGGAAAGACU 1787- A- 3455 UGUAUCAGUCUUUCC 1785- 122297 228232 GAUACA 1807 2282330. UGGUGAGA 1807 5.1 9.1 1 AD- A- 3007 CACCAGGAAAGACUG 1788- A- 3456 UUGUAUCAGUCUUUC 1786- 122297 228233 AUACAA 1808 2282332. CUGGUGAG 1808 6.1 1.1 1 AD- A- 3008 ACCAGGAAAGACUGA 1789- A- 3457 UCUGUAUCAGUCUUU 1787- 122297 228233 UACAGA 1809 2282334. CCUGGUGA 1809 7.1 3.1 1 AD- A- 3009 CCAGGAAAGACUGAU 1790- A- 3458 UUCUGUAUCAGUCUU 1788- 122297 228233 ACAGAA 1810 2282336. UCCUGGUG 1810 8.1 5.1 1 AD- A- 3010 CAGGAAAGACUGAU 1791- A- 3459 UUUCUGUAUCAGUCU 1789- 122297 228233 ACAGAAA 1811 2282338. UUCCUGGU 1811 9.1 7.1 1 AD- A- 3011 AGGAAAGACUGAUA 1792- A- 3460 UGUUCUGUAUCAGUC 1790- 122298 228233 CAGAACA 1812 2282340. UUUCCUGG 1812 0.1 9.1 1 AD- A- 3012 GGAAAGACUGAUAC 1793- A- 3461 UCGUUCUGUAUCAGU 1791- 122298 228234 AGAACGA 1813 2282342. CUUUCCUG 1813 1.1 1.1 1 AD- A- 3013 GAAAGACUGAUACA 1794- A- 3462 UUCGUUCUGUAUCAG 1792- 122298 228234 GAACGAA 1814 2282344. UCUUUCCU 1814 2.1 3.1 1 AD- A- 3014 AAAGACUGAUACAG 1795- A- 3463 UAUCGUUCUGUAUCA 1793- 122298 228234 AACGAUA 1815 2282346. GUCUUUCC 1815 3.1 5.1 1 AD- A- 3015 AAGACUGAUACAGA 1796- A- 3464 UGAUCGUUCUGUAUC 1794- 122298 228234 ACGAUCA 1816 2282348. AGUCUUUC 1816 4.1 7.1 1 AD- A- 3016 AGACUGAUACAGAAC 1797- A- 3465 UCGAUCGUUCUGUAU 1795- 122298 228234 GAUCGA 1817 2282350. CAGUCUUU 1817 5.1 9.1 1 AD- A- 3017 GACUGAUACAGAACG 1798- A- 3466 UUCGAUCGUUCUGUA 1796- 122298 228235 AUCGAA 1818 2282352. UCAGUCUU 1818 6.1 1.1 1 AD- A- 3018 ACUGAUACAGAACGA 1799- A- 3467 UAUCGAUCGUUCUGU 1797- 122298 228235 UCGAUA 1819 2282354. AUCAGUCU 1819 7.1 3.1 1 AD- A- 3019 CUGAUACAGAACGAU 1800- A- 3468 UUAUCGAUCGUUCUG 1798- 122298 228235 CGAUAA 1820 2282356. UAUCAGUC 1820 8.1 5.1 1 AD- A- 3020 UGAUACAGAACGAUC 1801- A- 3469 UGUAUCGAUCGUUCU 1799- 122298 228235 GAUACA 1821 2282358. GUAUCAGU 1821 9.1 7.1 1 AD- A- 3021 GAUACAGAACGAUCG 1802- A- 3470 UUGUAUCGAUCGUUC 1800- 122299 228235 AUACAA 1822 2282360. UGUAUCAG 1822 0.1 9.1 1 AD- A- 3022 AUACAGAACGAUCGA 1803- A- 3471 UCUGUAUCGAUCGUU 1801- 122299 228236 UACAGA 1823 2282362. CUGUAUCA 1823 1.1 1.1 1 AD- A- 3023 UACAGAACGAUCGAU 1804- A- 3472 UUCUGUAUCGAUCGU 1802- 122299 228236 ACAGAA 1824 2282364. UCUGUAUC 1824 2.1 3.1 1 AD- A- 3024 ACAGAACGAUCGAUA 1805- A- 3473 UUUCUGUAUCGAUCG 1803- 122299 228236 CAGAAA 1825 2282366. UUCUGUAU 1825 3.1 5.1 1 AD- A- 3025 CAGAACGAUCGAUAC 1806- A- 3474 UUUUCUGUAUCGAUC 1804- 122299 228236 AGAAAA 1826 2282368. GUUCUGUA 1826 4.1 7.1 1 AD- A- 3026 AGAACGAUCGAUACA 1807- A- 3475 UGUUUCUGUAUCGAU 1805- 122299 228236 GAAACA 1827 2282370. CGUUCUGU 1827 5.1 9.1 1 AD- A- 3027 GAACGAUCGAUACAG 1808- A- 3476 UGGUUUCUGUAUCGA 1806- 122299 228237 AAACCA 1828 2282372. UCGUUCUG 1828 6.1 1.1 1 AD- A- 3028 AACGAUCGAUACAGA 1809- A- 3477 UUGGUUUCUGUAUCG 1807- 122299 228237 AACCAA 1829 2282374. AUCGUUCU 1829 7.1 3.1 1 AD- A- 3029 ACGAUCGAUACAGAA 1810- A- 3478 UGUGGUUUCUGUAUC 1808- 122299 228237 ACCACA 1830 2282376. GAUCGUUC 1830 8.1 5.1 1 AD- A- 3030 CGAUCGAUACAGAAA 1811- A- 3479 UCGUGGUUUCUGUAU 1809- 122299 228237 CCACGA 1831 2282378. CGAUCGUU 1831 9.1 7.1 1 AD- A- 3031 CACCAUCACCAUCGA 1843- A- 3480 UUUCUGUCGAUGGUG 1841- 122300 228237 CAGAAA 1863 2282380. AUGGUGUG 1863 0.1 9.1 1 AD- A- 3032 CAUCACCAUCGACAG 1846- A- 3481 UCUGUUCUGUCGAUG 1844- 122300 228238 AACAGA 1866 2282382. GUGAUGGU 1866 1.1 1.1 1 AD- A- 3033 UCACCAUCGACAGAA 1848- A- 3482 UGACUGUUCUGUCGA 1846- 122300 228238 CAGUCA 1868 2282384. UGGUGAUG 1868 2.1 3.1 1 AD- A- 3034 CACCAUCGACAGAAC 1849- A- 3483 UGGACUGUUCUGUCG 1847- 122300 228238 AGUCCA 1869 2282386. AUGGUGAU 1869 3.1 5.1 1 AD- A- 3035 ACCAUCGACAGAACA 1850- A- 3484 UAGGACUGUUCUGUC 1848- 122300 228238 GUCCUA 1870 2282388. GAUGGUGA 1870 4.1 7.1 1 AD- A- 3036 CCAUCGACAGAACAG 1851- A- 3485 UAAGGACUGUUCUGU 1849- 122300 228238 UCCUUA 1871 2282390. CGAUGGUG 1871 5.1 9.1 1 AD- A- 3037 CAUCGACAGAACAGU 1852- A- 3486 UUAAGGACUGUUCUG 1850- 122300 228239 CCUUAA 1872 2282392. UCGAUGGU 1872 6.1 1.1 1 AD- A- 3038 AUCGACAGAACAGUC 1853- A- 3487 UUUAAGGACUGUUCU 1851- 122300 228239 CUUAAA 1873 2282394. GUCGAUGG 1873 7.1 3.1 1 AD- A- 3039 UCGACAGAACAGUCC 1854- A- 3488 UAUUAAGGACUGUUC 1852- 122300 228239 UUAAUA 1874 2282396. UGUCGAUG 1874 8.1 5.1 1 AD- A- 3040 CGACAGAACAGUCCU 1855- A- 3489 UGAUUAAGGACUGUU 1853- 122300 228239 UAAUCA 1875 2282398. CUGUCGAU 1875 9.1 7.1 1 AD- A- 3041 GACAGAACAGUCCUU 1856- A- 3490 UGGAUUAAGGACUGU 1854- 122301 228239 AAUCCA 1876 2282400. UCUGUCGA 1876 0.1 9.1 1 AD- A- 3042 ACAGAACAGUCCUUA 1857- A- 3491 UUGGAUUAAGGACUG 1855- 122301 228240 AUCCAA 1877 2282402. UUCUGUCG 1877 1.1 1.1 1 AD- A- 3043 AGAACAGUCCUUAAU 1859- A- 3492 UUCUGGAUUAAGGAC 1857- 122301 228240 CCAGAA 1879 2282404. UGUUCUGU 1879 2.1 3.1 1 AD- A- 3044 GAACAGUCCUUAAUC 1860- A- 3493 UUUCUGGAUUAAGGA 1858- 122301 228240 CAGAAA 1880 2282406. CUGUUCUG 1880 3.1 5.1 1 AD- A- 3045 AACAGUCCUUAAUCC 1861- A- 3494 UUUUCUGGAUUAAGG 1859- 122301 228240 AGAAAA 1881 2282408. ACUGUUCU 1881 4.1 7.1 1 AD- A- 3046 ACAGUCCUUAAUCCA 1862- A- 3495 UGUUUCUGGAUUAAG 1860- 122301 228240 GAAACA 1882 2282410. GACUGUUC 1882 5.1 9.1 1 AD- A- 3047 CAGUCCUUAAUCCAG 1863- A- 3496 UGGUUUCUGGAUUAA 1861- 122301 228241 AAACCA 1883 2282412. GGACUGUU 1883 6.1 1.1 1 AD- A- 3048 AGUCCUUAAUCCAGA 1864- A- 3497 UAGGUUUCUGGAUUA 1862- 122301 228241 AACCUA 1884 2282414. AGGACUGU 1884 7.1 3.1 1 AD- A- 3049 GUCCUUAAUCCAGAA 1865- A- 3498 UCAGGUUUCUGGAUU 1863- 122301 228241 ACCUGA 1885 2282416. AAGGACUG 1885 8.1 5.1 1 AD- A- 3050 UCCUUAAUCCAGAAA 1866- A- 3499 UUCAGGUUUCUGGAU 1864- 122301 228241 CCUGAA 1886 2282418. UAAGGACU 1886 9.1 7.1 1 AD- A- 3051 CCUUAAUCCAGAAAC 1867- A- 3500 UUUCAGGUUUCUGGA 1865- 122302 228241 CUGAAA 1887 2282420. UUAAGGAC 1887 0.1 9.1 1 AD- A- 3052 CUUAAUCCAGAAACC 1868- A- 3501 UUUUCAGGUUUCUGG 1866- 122302 228242 UGAAAA 1888 2282422. AUUAAGGA 1888 1.1 1.1 1 AD- A- 3053 UUAAUCCAGAAACCU 1869- A- 3502 UAUUUCAGGUUUCUG 1867- 122302 228242 GAAAUA 1889 2282424. GAUUAAGG 1889 2.1 3.1 1 AD- A- 3054 CAGAAACCUGAAAUG 1875- A- 3503 UUCCUUCAUUUCAGG 1873- 122302 228242 AAGGAA 1895 2282426. UUUCUGGA 1895 3.1 5.1 1 AD- A- 3055 AGAAACCUGAAAUG 1876- A- 3504 UUUCCUUCAUUUCAG 1874- 122302 228242 AAGGAAA 1896 2282428. GUUUCUGG 1896 4.1 7.1 1 AD- A- 3056 GAAACCUGAAAUGA 1877- A- 3505 UCUUCCUUCAUUUCA 1875- 122302 228242 AGGAAGA 1897 2282430. GGUUUCUG 1897 5.1 9.1 1 AD- A- 3057 AAACCUGAAAUGAA 1878- A- 3506 UUCUUCCUUCAUUUC 1876- 122302 228243 GGAAGAA 1898 2282432. AGGUUUCU 1898 6.1 1.1 1 AD- A- 3058 AACCUGAAAUGAAG 1879- A- 3507 UCUCUUCCUUCAUUU 1877- 122302 228243 GAAGAGA 1899 2282434. CAGGUUUC 1899 7.1 3.1 1 AD- A- 3059 CCUGAAAUGAAGGA 1881- A- 3508 UUCCUCUUCCUUCAU 1879- 122302 228243 AGAGGAA 1901 2282436. UUCAGGUU 1901 8.1 5.1 1 AD- A- 3060 AUGAAGGAAGAGGA 1887- A- 3509 UAGAGUCUCCUCUUC 1885- 122302 228243 GACUCUA 1907 2282438. CUUCAUUU 1907 9.1 7.1 1 AD- A- 3061 UCCCUCUUGGAAUUG 1977- A- 3510 UGAAUCCAAUUCCAA 1975- 122303 228243 GAUUCA 1997 2282440. GAGGGACC 1997 0.1 9.1 1 AD- A- 3062 CCUCUUGGAAUUGGA 1979- A- 3511 UGCGAAUCCAAUUCC 1977- 122303 228244 UUCGCA 1999 2282442. AAGAGGGA 1999 1.1 1.1 1 AD- A- 3063 CUCUUGGAAUUGGA 1980- A- 3512 UGGCGAAUCCAAUUC 1978- 122303 228244 UUCGCCA 2000 2282444. CAAGAGGG 2000 2.1 3.1 1 AD- A- 3064 UUGGAAUUGGAUUC 1983- A- 3513 UAAUGGCGAAUCCAA 1981- 122303 228244 GCCAUUA 2003 2282446. UUCCAAGA 2003 3.1 5.1 1 AD- A- 3065 UGGAAUUGGAUUCG 1984- A- 3514 UAAAUGGCGAAUCCA 1982- 122303 228244 CCAUUUA 2004 2282448. AUUCCAAG 2004 4.1 7.1 1 AD- A- 3066 GGAAUUGGAUUCGCC 1985- A- 3515 UAAAAUGGCGAAUCC 1983- 122303 228244 AUUUUA 2005 2282450. AAUUCCAA 2005 5.1 9.1 1 AD- A- 3067 GAAUUGGAUUCGCCA 1986- A- 3516 UUAAAAUGGCGAAUC 1984- 122303 228245 UUUUAA 2006 2282452. CAAUUCCA 2006 6.1 1.1 1 AD- A- 3068 AAUUGGAUUCGCCAU 1987- A- 3517 UAUAAAAUGGCGAAU 1985- 122303 228245 UUUAUA 2007 2282454. CCAAUUCC 2007 7.1 3.1 1 AD- A- 3069 AUUGGAUUCGCCAUU 1988- A- 3518 UAAUAAAAUGGCGAA 1986- 122303 228245 UUAUUA 2008 2282456. UCCAAUUC 2008 8.1 5.1 1 AD- A- 3070 UUGGAUUCGCCAUUU 1989- A- 3519 UAAAUAAAAUGGCGA 1987- 122303 228245 UAUUUA 2009 2282458. AUCCAAUU 2009 9.1 7.1 1 AD- A- 3071 UGGAUUCGCCAUUUU 1990- A- 3520 UAAAAUAAAAUGGCG 1988- 122304 228245 AUUUUA 2010 2282460. AAUCCAAU 2010 0.1 9.1 1 AD- A- 3072 GGAUUCGCCAUUUUA 1991- A- 3521 UAAAAAUAAAAUGGC 1989- 122304 228246 UUUUUA 2011 2282462. GAAUCCAA 2011 1.1 1.1 1 AD- A- 3073 GAUUCGCCAUUUUAU 1992- A- 3522 UGAAAAAUAAAAUGG 1990- 122304 228246 UUUUCA 2012 2282464. CGAAUCCA 2012 2.1 3.1 1 AD- A- 3074 UCGCCAUUUUAUUUU 1995- A- 3523 UCAAGAAAAAUAAAA 1993- 122304 228246 UCUUGA 2015 2282466. UGGCGAAU 2015 3.1 5.1 1 AD- A- 3075 CGCCAUUUUAUUUUU 1996- A- 3524 UGCAAGAAAAAUAAA 1994- 122304 228246 CUUGCA 2016 2282468. AUGGCGAA 2016 4.1 7.1 1 AD- A- 3076 GCCAUUUUAUUUUUC 1997- A- 3525 UAGCAAGAAAAAUAA 1995- 122304 228246 UUGCUA 2017 2282470. AAUGGCGA 2017 5.1 9.1 1 AD- A- 3077 CCAUUUUAUUUUUCU 1998- A- 3526 UCAGCAAGAAAAAUA 1996- 122304 228247 UGCUGA 2018 2282472. AAAUGGCG 2018 6.1 1.1 1 AD- A- 3078 CAUUUUAUUUUUCU 1999- A- 3527 UGCAGCAAGAAAAAU 1997- 122304 228247 UGCUGCA 2019 2282474. AAAAUGGC 2019 7.1 3.1 1 AD- A- 3079 AUUUUAUUUUUCUU 2000- A- 3528 UAGCAGCAAGAAAAA 1998- 122304 228247 GCUGCUA 2020 2282476. UAAAAUGG 2020 8.1 5.1 1 AD- A- 3080 UUUUAUUUUUCUUG 2001- A- 3529 UUAGCAGCAAGAAAA 1999- 122304 228247 CUGCUAA 2021 2282478. AUAAAAUG 2021 9.1 7.1 1 AD- A- 3081 UUUCUUGCUGCUAAA 2008- A- 3530 UGGUGAUUUAGCAGC 2006- 122305 228247 UCACCA 2028 2282480. AAGAAAAA 2028 0.1 9.1 1 AD- A- 3082 UCACCGAGCCCGGAA 2023- A- 3531 UUAAUCUUCCGGGCU 2021- 122305 228248 GAUUAA 2043 2282482. CGGUGAUU 2043 1.1 1.1 1 AD- A- 3083 GCCCGGAAGAUUAGA 2030- A- 3532 UAACUCUCUAAUCUU 2028- 122305 228248 GAGUUA 2050 2282484. CCGGGCUC 2050 2.1 3.1 1 AD- A- 3084 CCCGGAAGAUUAGAG 2031- A- 3533 UAAACUCUCUAAUCU 2029- 122305 228248 AGUUUA 2051 2282486. UCCGGGCU 2051 3.1 5.1 1 AD- A- 3085 CGGAAGAUUAGAGA 2033- A- 3534 UUAAAACUCUCUAAU 2031- 122305 228248 GUUUUAA 2053 2282488. CUUCCGGG 2053 4.1 7.1 1 AD- A- 3086 GGAAGAUUAGAGAG 2034- A- 3535 UAUAAAACUCUCUAA 2032- 122305 228248 UUUUAUA 2054 2282490. UCUUCCGG 2054 5.1 9.1 1 AD- A- 3087 GAAGAUUAGAGAGU 2035- A- 3536 UAAUAAAACUCUCUA 2033- 122305 228249 UUUAUUA 2055 2282492. AUCUUCCG 2055 6.1 1.1 1 AD- A- 3088 AAGAUUAGAGAGUU 2036- A- 3537 UAAAUAAAACUCUCU 2034- 122305 228249 UUAUUUA 2056 2282494. AAUCUUCC 2056 7.1 3.1 1 AD- A- 3089 AGAUUAGAGAGUUU 2037- A- 3538 UGAAAUAAAACUCUC 2035- 122305 228249 UAUUUCA 2057 2282496. UAAUCUUC 2057 8.1 5.1 1 AD- A- 3090 AUUAGAGAGUUUUA 2039- A- 3539 UCAGAAAUAAAACUC 2037- 122305 228249 UUUCUGA 2059 2282498. UCUAAUCU 2059 9.1 7.1 1 AD- A- 3091 UUAGAGAGUUUUAU 2040- A- 3540 UCCAGAAAUAAAACU 2038- 122306 228249 UUCUGGA 2060 2282500. CUCUAAUC 2060 0.1 9.1 1 AD- A- 3092 UAGAGAGUUUUAUU 2041- A- 3541 UCCCAGAAAUAAAAC 2039- 122306 228250 UCUGGGA 2061 2282502. UCUCUAAU 2061 1.1 1.1 1 AD- A- 3093 AGAGAGUUUUAUUU 2042- A- 3542 UUCCCAGAAAUAAAA 2040- 122306 228250 CUGGGAA 2062 2282504. CUCUCUAA 2062 2.1 3.1 1 AD- A- 3094 GAGAGUUUUAUUUC 2043- A- 3543 UAUCCCAGAAAUAAA 2041- 122306 228250 UGGGAUA 2063 2282506. ACUCUCUA 2063 3.1 5.1 1 AD- A- 3095 AGAGUUUUAUUUCU 2044- A- 3544 UAAUCCCAGAAAUAA 2042- 122306 228250 GGGAUUA 2064 2282508. AACUCUCU 2064 4.1 7.1 1 AD- A- 3096 GAGUUUUAUUUCUG 2045- A- 3545 UGAAUCCCAGAAAUA 2043- 122306 228250 GGAUUCA 2065 2282510. AAACUCUC 2065 5.1 9.1 1 AD- A- 3097 AGUUUUAUUUCUGG 2046- A- 3546 UGGAAUCCCAGAAAU 2044- 122306 228251 GAUUCCA 2066 2282512. AAAACUCU 2066 6.1 1.1 1 AD- A- 3098 GUUUUAUUUCUGGG 2047- A- 3547 UAGGAAUCCCAGAAA 2045- 122306 228251 AUUCCUA 2067 2282514. UAAAACUC 2067 7.1 3.1 1 AD- A- 3099 UUUUAUUUCUGGGA 2048- A- 3548 UCAGGAAUCCCAGAA 2046- 122306 228251 UUCCUGA 2068 2282516. AUAAAACU 2068 8.1 5.1 1 AD- A- 3100 UUUAUUUCUGGGAU 2049- A- 3549 UACAGGAAUCCCAGA 2047- 122306 228251 UCCUGUA 2069 2282518. AAUAAAAC 2069 9.1 7.1 1 AD- A- 3101 UUAUUUCUGGGAUU 2050- A- 3550 UUACAGGAAUCCCAG 2048- 122307 228251 CCUGUAA 2070 2282520. AAAUAAAA 2070 0.1 9.1 1 AD- A- 3102 UAUUUCUGGGAUUCC 2051- A- 3551 UCUACAGGAAUCCCA 2049- 122307 228252 UGUAGA 2071 2282522. GAAAUAAA 2071 1.1 1.1 1 AD- A- 3103 AUUUCUGGGAUUCCU 2052- A- 3552 UUCUACAGGAAUCCC 2050- 122307 228252 GUAGAA 2072 2282524. AGAAAUAA 2072 2.1 3.1 1 AD- A- 3104 UUUCUGGGAUUCCUG 2053- A- 3553 UGUCUACAGGAAUCC 2051- 122307 228252 UAGACA 2073 2282526. CAGAAAUA 2073 3.1 5.1 1 AD- A- 3105 UCUGGGAUUCCUGUA 2055- A- 3554 UGUGUCUACAGGAAU 2053- 122307 228252 GACACA 2075 2282528. CCCAGAAA 2075 4.1 7.1 1 AD- A- 3106 CUGGGAUUCCUGUAG 2056- A- 3555 UUGUGUCUACAGGAA 2054- 122307 228252 ACACAA 2076 2282530. UCCCAGAA 2076 5.1 9.1 1 AD- A- 3107 UGGGAUUCCUGUAG 2057- A- 3556 UGUGUGUCUACAGGA 2055- 122307 228253 ACACACA 2077 2282532. AUCCCAGA 2077 6.1 1.1 1 AD- A- 3108 GGGAUUCCUGUAGAC 2058- A- 3557 UGGUGUGUCUACAGG 2056- 122307 228253 ACACCA 2078 2282534. AAUCCCAG 2078 7.1 3.1 1 AD- A- 3109 ACACACCCACCCACA 2071- A- 3558 UAUGUAUGUGGGUGG 2069- 122307 228253 UACAUA 2091 2282536. GUGUGUCU 2091 8.1 5.1 1 AD- A- 3110 ACCCACCCACAUACA 2075- A- 3559 UAUGUAUGUAUGUGG 2073- 122307 228253 UACAUA 2095 2282538. GUGGGUGU 2095 9.1 7.1 1 AD- A- 3111 CCCACCCACAUACAU 2076- A- 3560 UAAUGUAUGUAUGUG 2074- 122308 228253 ACAUUA 2096 2282540. GGUGGGUG 2096 0.1 9.1 1 AD- A- 3112 CCACCCACAUACAUA 2077- A- 3561 UAAAUGUAUGUAUGU 2075- 122308 228254 CAUUUA 2097 2282542. GGGUGGGU 2097 1.1 1.1 1 AD- A- 3113 CACCCACAUACAUAC 2078- A- 3562 UUAAAUGUAUGUAUG 2076- 122308 228254 AUUUAA 2098 2282544. UGGGUGGG 2098 2.1 3.1 1 AD- A- 3114 CCACAUACAUACAUU 2081- A- 3563 UAUAUAAAUGUAUGU 2079- 122308 228254 UAUAUA 2101 2282546. AUGUGGGU 2101 3.1 5.1 1 AD- A- 3115 CACAUACAUACAUUU 2082- A- 3564 UUAUAUAAAUGUAUG 2080- 122308 228254 AUAUAA 2102 2282548. UAUGUGGG 2102 4.1 7.1 1 AD- A- 3116 UUAAAUUAACAGUG 2171- A- 3565 UCAUUAGCACUGUUA 2169- 122308 228254 CUAAUGA 2191 2282550. AUUUAAAA 2191 5.1 9.1 1 AD- A- 3117 UAAAUUAACAGUGC 2172- A- 3566 UACAUUAGCACUGUU 2 nO- 122308 228255 UAAUGUA 2192 2282552. AAUUUAAA 2192 6.1 1.1 1 AD- A- 3118 AAAUUAACAGUGCU 2173- A- 3567 UAACAUUAGCACUGU 2171- 122308 228255 AAUGUUA 2193 2282554. UAAUUUAA 2193 7.1 3.1 1 AD- A- 3119 AAUUAACAGUGCUA 2174- A- 3568 UUAACAUUAGCACUG 2172- 122308 228255 AUGUUAA 2194 2282556. UUAAUUUA 2194 8.1 5.1 1 AD- A- 3120 AUUAACAGUGCUAA 2175- A- 3569 UAUAACAUUAGCACU 2173- 122308 228255 UGUUAUA 2195 2282558. GUUAAUUU 2195 9.1 7.1 1 AD- A- 3121 UUAACAGUGCUAAU 2176- A- 3570 UAAUAACAUUAGCAC 2174- 122309 228255 GUUAUUA 2196 2282560. UGUUAAUU 2196 0.1 9.1 1 AD- A- 3122 UAACAGUGCUAAUG 2177- A- 3571 UCAAUAACAUUAGCA 2175- 122309 228256 UUAUUGA 2197 2282562. CUGUUAAU 2197 1.1 1.1 1 AD- A- 3123 AACAGUGCUAAUGU 2178- A- 3572 UCCAAUAACAUUAGC 2176- 122309 228256 UAUUGGA 2198 2282564. ACUGUUAA 2198 2.1 3.1 1 AD- A- 3124 ACAGUGCUAAUGUU 2179- A- 3573 UACCAAUAACAUUAG 2177- 122309 228256 AUUGGUA 2199 2282566. CACUGUUA 2199 3.1 5.1 1 AD- A- 3125 CAGUGCUAAUGUUA 2180- A- 3574 UCACCAAUAACAUUA 2178- 122309 228256 UUGGUGA 2200 2282568. GCACUGUU 2200 4.1 7.1 1 AD- A- 3126 GUGCUAAUGUUAUU 2182- A- 3575 UGACACCAAUAACAU 2180- 122309 228256 GGUGUCA 2202 2282570. UAGCACUG 2202 5.1 9.1 1 AD- A- 3127 UGCUAAUGUUAUUG 2183- A- 3576 UAGACACCAAUAACA 2181- 122309 228257 GUGUCUA 2203 2282572. UUAGCACU 2203 6.1 1.1 1 AD- A- 3128 GCUAAUGUUAUUGG 2184- A- 3577 UAAGACACCAAUAAC 2182- 122309 228257 UGUCUUA 2204 2282574. AUUAGCAC 2204 7.1 3.1 1 AD- A- 3129 CUAAUGUUAUUGGU 2185- A- 3578 UGAAGACACCAAUAA 2183- 122309 228257 GUCUUCA 2205 2282576. CAUUAGCA 2205 8.1 5.1 1 AD- A- 3130 UAAUGUUAUUGGUG 2186- A- 3579 UUGAAGACACCAAUA 2184- 122309 228257 UCUUCAA 2206 2282578. ACAUUAGC 2206 9.1 7.1 1 AD- A- 3131 AAUGUUAUUGGUGU 2187- A- 3580 UGUGAAGACACCAAU 2185- 122310 228257 CUUCACA 2207 2282580. AACAUUAG 2207 0.1 9.1 1 AD- A- 3132 AUGUUAUUGGUGUC 2188- A- 3581 UAGUGAAGACACCAA 2186- 122310 228258 UUCACUA 2208 2282582. UAACAUUA 2208 1.1 1.1 1 AD- A- 3133 UGUUAUUGGUGUCU 2189- A- 3582 UCAGUGAAGACACCA 2187- 122310 228258 UCACUGA 2209 2282584. AUAACAUU 2209 2.1 3.1 1 AD- A- 3134 GUUAUUGGUGUCUU 2190- A- 3583 UCCAGUGAAGACACC 2188- 122310 228258 CACUGGA 2210 2282586. AAUAACAU 2210 3.1 5.1 1 AD- A- 3135 UUAUUGGUGUCUUC 2191- A- 3584 UUCCAGUGAAGACAC 2189- 122310 228258 ACUGGAA 2211 2282588. CAAUAACA 2211 4.1 7.1 1 AD- A- 3136 UGGUGUCUUCACUGG 2195- A- 3585 UUACAUCCAGUGAAG 2193- 122310 228258 AUGUAA 2215 2282590. ACACCAAU 2215 5.1 9.1 1 AD- A- 3137 UGUCUUCACUGGAUG 2198- A- 3586 UAAAUACAUCCAGUG 2196- 122310 228259 UAUUUA 2218 2282592. AAGACACC 2218 6.1 1.1 1 AD- A- 3138 CACUGGAUGUAUUU 2204- A- 3587 UGCAGUCAAAUACAU 2202- 122310 228259 GACUGCA 2224 2282594. CCAGUGAA 2224 7.1 3.1 1 AD- A- 3139 ACUGGAUGUAUUUG 2205- A- 3588 UAGCAGUCAAAUACA 2203- 122310 228259 ACUGCUA 2225 2282596. UCCAGUGA 2225 8.1 5.1 1 AD- A- 3140 UGGAUGUAUUUGAC 2207- A- 3589 UACAGCAGUCAAAUA 2205- 122310 228259 UGCUGUA 2227 2282598. CAUCCAGU 2227 9.1 7.1 1 AD- A- 3141 UAUUUGACUGCUGU 2213- A- 3590 UAAGUCCACAGCAGU 2211- 122311 228259 GGACUUA 2233 2282600. CAAAUACA 2233 0.1 9.1 1 AD- A- 3142 UUUGACUGCUGUGG 2215- A- 3591 UUCAAGUCCACAGCA 2213- 122311 228260 ACUUGAA 2235 2282602. GUCAAAUA 2235 1.1 1.1 1 AD- A- 3143 GCUGUGGACUUGAG 2222- A- 3592 UUCCCAACUCAAGUCC 2220- 122311 228260 UUGGGAA 2242 2282604. ACAGCAG 2242 2.1 3.1 1 AD- A- 3144 UCCCACUCAGAUCCU 2251- A- 3593 UCUGUCAGGAUCUGA 2249- 122311 228260 GACAGA 2271 2282606. GUGGGAAC 2271 3.1 5.1 1 AD- A- 3145 CCCACUCAGAUCCUG 2252- A- 3594 UCCUGUCAGGAUCUG 2250- 122311 228260 ACAGGA 2272 2282608. AGUGGGAA 2272 4.1 7.1 1 AD- A- 3146 GGAGGAGAUGAGAG 2277- A- 3595 UCAGAGUCUCUCAUC 2275- 122311 228260 ACUCUGA 2297 2282610. UCCUCCUC 2297 5.1 9.1 1 AD- A- 3147 GAGGAGAUGAGAGA 2278- A- 3596 UCCAGAGUCUCUCAU 2276- 122311 228261 CUCUGGA 2298 2282612. CUCCUCCU 2298 6.1 1.1 1 AD- A- 3148 GGAGAUGAGAGACU 2280- A- 3597 UUGCCAGAGUCUCUC 2278- 122311 228261 CUGGCAA 2300 2282614. AUCUCCUC 2300 7.1 3.1 1 AD- A- 3149 GAGACUCUGGCAUGA 2288- A- 3598 UAAAGAUCAUGCCAG 2286- 122311 228261 UCUUUA 2308 2282616. AGUCUCUC 2308 8.1 5.1 1 AD- A- 3150 AGACUCUGGCAUGAU 2289- A- 3599 UAAAAGAUCAUGCCA 2287- 122311 228261 CUUUUA 2309 2282618. GAGUCUCU 2309 9.1 7.1 1 AD- A- 3151 UUUUGGGAACACCGA 2392- A- 3600 UGUUUGUCGGUGUUC 2390- 122312 228261 CAAACA 2412 2282620. CCAAAACU 2412 0.1 9.1 1 AD- A- 3152 GAGCUUCAGGACAUU 2511- A- 3601 UACAGCAAUGUCCUG 2509- 122312 228262 GCUGUA 2531 2282622. AAGCUCCC 2531 1.1 1.1 1 AD- A- 3153 GCUUCAGGACAUUGC 2513- A- 3602 UGCACAGCAAUGUCC 2511- 122312 228262 UGUGCA 2533 2282624. UGAAGCUC 2533 2.1 3.1 1 AD- A- 3154 CUUCAGGACAUUGCU 2514- A- 3603 UAGCACAGCAAUGUC 2512- 122312 228262 GUGCUA 2534 2282626. CUGAAGCU 2534 3.1 5.1 1 AD- A- 3155 UUCAGGACAUUGCUG 2515- A- 3604 UAAGCACAGCAAUGU 2513- 122312 228262 UGCUUA 2535 2282628. CCUGAAGC 2535 4.1 7.1 1 AD- A- 3156 UCAGGACAUUGCUGU 2516- A- 3605 UAAAGCACAGCAAUG 2514- 122312 228262 GCUUUA 2536 2282630. UCCUGAAG 2536 5.1 9.1 1 AD- A- 3157 CAGGACAUUGCUGUG 2517- A- 3606 UCAAAGCACAGCAAU 2515- 122312 228263 CUUUGA 2537 2282632. GUCCUGAA 2537 6.1 1.1 1 AD- A- 3158 CGCUUACUCUCACCU 2615- A- 3607 UGAAGCAGGUGAGAG 2613- 122312 228263 GCUUCA 2635 2282634. UAAGCGAA 2635 7.1 3.1 1 AD- A- 3159 GCUUACUCUCACCUG 2616- A- 3608 UAGAAGCAGGUGAGA 2614- 122312 228263 CUUCUA 2636 2282636. GUAAGCGA 2636 8.1 5.1 1 AD- A- 3160 UUACUCUCACCUGCU 2618- A- 3609 UUCAGAAGCAGGUGA 2616- 122312 228263 UCUGAA 2638 2282638. GAGUAAGC 2638 9.1 7.1 1 AD- A- 3161 CUCUCACCUGCUUCU 2621- A- 3610 UAACUCAGAAGCAGG 2619- 122313 228263 GAGUUA 2641 2282640. UGAGAGUA 2641 0.1 9.1 1 AD- A- 3162 UCUCACCUGCUUCUG 2622- A- 3611 UCAACUCAGAAGCAG 2620- 122313 228264 AGUUGA 2642 2282642. GUGAGAGU 2642 1.1 1.1 1 AD- A- 3163 CUCACCUGCUUCUGA 2623- A- 3612 UGCAACUCAGAAGCA 2621- 122313 228264 GUUGCA 2643 2282644. GGUGAGAG 2643 2.1 3.1 1 AD- A- 3164 UCACCUGCUUCUGAG 2624- A- 3613 UGGCAACUCAGAAGC 2622- 122313 228264 UUGCCA 2644 2282646. AGGUGAGA 2644 3.1 5.1 1 AD- A- 3165 CACCUGCUUCUGAGU 2625- A- 3614 UGGGCAACUCAGAAG 2623- 122313 228264 UGCCCA 2645 2282648. CAGGUGAG 2645 4.1 7.1 1 AD- A- 3166 ACCUGCUUCUGAGUU 2626- A- 3615 UUGGGCAACUCAGAA 2624- 122313 228264 GCCCAA 2646 2282650. GCAGGUGA 2646 5.1 9.1 1 AD- A- 3167 CCUGCUUCUGAGUUG 2627- A- 3616 UCUGGGCAACUCAGA 2625- 122313 228265 CCCAGA 2647 2282652. AGCAGGUG 2647 6.1 1.1 1 AD- A- 3168 CGGCGAAGAGAAGA 2667- A- 3617 UUGUGUCUCUUCUCU 2665- 122313 228265 GACACAA 2687 2282654. UCGCCGGG 2687 7.1 3.1 1 AD- A- 3169 GGCGAAGAGAAGAG 2668- A- 3618 UAUGUGUCUCUUCUC 2666- 122313 228265 ACACAUA 2688 2282656. UUCGCCGG 2688 8.1 5.1 1 AD- A- 3170 GCGAAGAGAAGAGA 2669- A- 3619 UAAUGUGUCUCUUCU 2667- 122313 228265 CACAUUA 2689 2282658. CUUCGCCG 2689 9.1 7.1 1 AD- A- 3171 CGAAGAGAAGAGAC 2670- A- 3620 UCAAUGUGUCUCUUC 2668- 122314 228265 ACAUUGA 2690 2282660. UCUUCGCC 2690 0.1 9.1 1 AD- A- 3172 GAAGAGAAGAGACA 2671- A- 3621 UACAAUGUGUCUCUU 2669- 122314 228266 CAUUGUA 2691 2282662. CUCUUCGC 2691 1.1 1.1 1 AD- A- 3173 AAGAGAAGAGACAC 2672- A- 3622 UAACAAUGUGUCUCU 2670- 122314 228266 AUUGUUA 2692 2282664. UCUCUUCG 2692 2.1 3.1 1 AD- A- 3174 AGAGAAGAGACACA 2673- A- 3623 UCAACAAUGUGUCUC 2671- 122314 228266 UUGUUGA 2693 2282666. uucucuuc 2693 3.1 5.1 1 AD- A- 3175 GAGAAGAGACACAU 2674- A- 3624 UCCAACAAUGUGUCU 2672- 122314 228266 UGUUGGA 2694 2282668. cuucucuu 2694 4.1 7.1 1 AD- A- 3176 AGAAGAGACACAUU 2675- A- 3625 UUCCAACAAUGUGUC 2673- 122314 228266 GUUGGAA 2695 2282670. UCUUCUCU 2695 5.1 9.1 1 AD- A- 3177 GAAGAGACACAUUG 2676- A- 3626 UUUCCAACAAUGUGU 2674- 122314 228267 UUGGAAA 2696 2282672. CUCUUCUC 2696 6.1 1.1 1 AD- A- 3178 AAGAGACACAUUGU 2677- A- 3627 UCUUCCAACAAUGUG 2675- 122314 228267 UGGAAGA 2697 2282674. UCUCUUCU 2697 7.1 3.1 1 AD- A- 3179 AGAGACACAUUGUU 2678- A- 3628 UUCUUCCAACAAUGU 2676- 122314 228267 GGAAGAA 2698 2282676. GUCUCUUC 2698 8.1 5.1 1 AD- A- 3180 GAGACACAUUGUUG 2679- A- 3629 UUUCUUCCAACAAUG 2677- 122314 228267 GAAGAAA 2699 2282678. UGUCUCUU 2699 9.1 7.1 1 AD- A- 3181 AGACACAUUGUUGG 2680- A- 3630 UCUUCUUCCAACAAU 2678- 122315 228267 AAGAAGA 2700 2282680. GUGUCUCU 2700 0.1 9.1 1 AD- A- 3182 GACACAUUGUUGGA 2681- A- 3631 UGCUUCUUCCAACAA 2679- 122315 228268 AGAAGCA 2701 2282682. UGUGUCUC 2701 1.1 1.1 1 AD- A- 3183 ACACAUUGUUGGAA 2682- A- 3632 UUGCUUCUUCCAACA 2680- 122315 228268 GAAGCAA 2702 2282684. AUGUGUCU 2702 2.1 3.1 1 AD- A- 3184 CACAUUGUUGGAAG 2683- A- 3633 UCUGCUUCUUCCAAC 2681- 122315 228268 AAGCAGA 2703 2282686. AAUGUGUC 2703 3.1 5.1 1 AD- A- 3185 UAUGUCCUCACACCA 2781- A- 3634 UUUCAAUGGUGUGAG 2779- 122315 228268 UUGAAA 2801 2282688. GACAUAGG 2801 4.1 7.1 1 AD- A- 3186 UCCUCACACCAUUGA 2785- A- 3635 UUGGUUUCAAUGGUG 2783- 122315 228268 AACCAA 2805 2282690. UGAGGACA 2805 5.1 9.1 1 AD- A- 3187 CCUCACACCAUUGAA 2786- A- 3636 UGUGGUUUCAAUGGU 2784- 122315 228269 ACCACA 2806 2282692. GUGAGGAC 2806 6.1 1.1 1 AD- A- 3188 CUCACACCAUUGAAA 2787- A- 3637 UAGUGGUUUCAAUGG 2785- 122315 228269 CCACUA 2807 2282694. UGUGAGGA 2807 7.1 3.1 1 AD- A- 3189 UCACACCAUUGAAAC 2788- A- 3638 UUAGUGGUUUCAAUG 2786- 122315 228269 CACUAA 2808 2282696. GUGUGAGG 2808 8.1 5.1 1 AD- A- 3190 CACACCAUUGAAACC 2789- A- 3639 UCUAGUGGUUUCAAU 2787- 122315 228269 ACUAGA 2809 2282698. GGUGUGAG 2809 9.1 7.1 1 AD- A- 3191 CCAUUGAAACCACUA 2793- A- 3640 UAGAACUAGUGGUUU 2791- 122316 228269 GUUCUA 2813 2282700. CAAUGGUG 2813 0.1 9.1 1 AD- A- 3192 CAUUGAAACCACUAG 2794- A- 3641 UCAGAACUAGUGGUU 2792- 122316 228270 UUCUGA 2814 2282702. UCAAUGGU 2814 1.1 1.1 1 AD- A- 3193 AUUGAAACCACUAGU 2795- A- 3642 UACAGAACUAGUGGU 2793- 122316 228270 UCUGUA 2815 2282704. UUCAAUGG 2815 2.1 3.1 1 AD- A- 3194 UUGAAACCACUAGUU 2796- A- 3643 UGACAGAACUAGUGG 2794- 122316 228270 CUGUCA 2816 2282706. UUUCAAUG 2816 3.1 5.1 1 AD- A- 3195 UGAAACCACUAGUUC 2797- A- 3644 UGGACAGAACUAGUG 2795- 122316 228270 UGUCCA 2817 2282708. GUUUCAAU 2817 4.1 7.1 1 AD- A- 3196 GACCUGGUUGUGUG 2825- A- 3645 UCACACACACACAACC 2823- 122316 228270 UGUGUGA 2845 2282710. AGGUCUC 2845 5.1 9.1 1 AD- A- 3197 ACCUGGUUGUGUGU 2826- A- 3646 UUCACACACACACAAC 2824- 122316 228271 GUGUGAA 2846 2282712. CAGGUCU 2846 6.1 1.1 1 AD- A- 3198 CCUGGUUGUGUGUG 2827- A- 3647 UCUCACACACACACAA 2825- 122316 228271 UGUGAGA 2847 2282714. CCAGGUC 2847 7.1 3.1 1 AD- A- 3199 CUGGUUGUGUGUGU 2828- A- 3648 UACUCACACACACACA 2826- 122316 228271 GUGAGUA 2848 2282716. ACCAGGU 2848 8.1 5.1 1 AD- A- 3200 UGGUUGUGUGUGUG 2829- A- 3649 UCACUCACACACACAC 2827- 122316 228271 UGAGUGA 2849 2282718. AACCAGG 2849 9.1 7.1 1 AD- A- 3201 GGUUGUGUGUGUGU 2830- A- 3650 UCCACUCACACACACA 2828- 122317 228271 GAGUGGA 2850 2282720. CAACCAG 2850 0.1 9.1 1 AD- A- 3202 GUGUGUGUGUGAGU 2834- A- 3651 UUCAACCACUCACACA 2832- 122317 228272 GGUUGAA 2854 2282722. CACACAA 2854 1.1 1.1 1 AD- A- 3203 UGUGUGUGUGAGUG 2835- A- 3652 UGUCAACCACUCACAC 2833- 122317 228272 GUUGACA 2855 2282724. ACACACA 2855 2.1 3.1 1 AD- A- 3204 UGUGUGUGAGUGGU 2837- A- 3653 UAGGUCAACCACUCA 2835- 122317 228272 UGACCUA 2857 2282726. CACACACA 2857 3.1 5.1 1 AD- A- 3205 UGUGUGAGUGGUUG 2839- A- 3654 UGAAGGUCAACCACU 2837- 122317 228272 ACCUUCA 2859 2282728. CACACACA 2859 4.1 7.1 1 AD- A- 3206 GUGUGAGUGGUUGA 2840- A- 3655 UGGAAGGUCAACCAC 2838- 122317 228272 CCUUCCA 2860 2282730. UCACACAC 2860 5.1 9.1 1 AD- A- 3207 UGUGAGUGGUUGAC 2841- A- 3656 UAGGAAGGUCAACCA 2839- 122317 228273 CUUCCUA 2861 2282732. CUCACACA 2861 6.1 1.1 1 AD- A- 3208 UGAGUGGUUGACCU 2843- A- 3657 UGGAGGAAGGUCAAC 2841- 122317 228273 UCCUCCA 2863 2282734. CACUCACA 2863 7.1 3.1 1 AD- A- 3209 GUGGUUGACCUUCCU 2846- A- 3658 UGAUGGAGGAAGGUC 2844- 122317 228273 CCAUCA 2866 2282736. AACCACUC 2866 8.1 5.1 1 AD- A- 3210 UUGUGGAGGCAGAG 2926- A- 3659 UUCUUUUCUCUGCCU 2924- 122317 228273 AAAAGAA 2946 2282738. CCACAAUG 2946 9.1 7.1 1 AD- A- 3211 UGUGGAGGCAGAGA 2927- A- 3660 UCUCUUUUCUCUGCC 2925- 122318 228273 AAAGAGA 2947 2282740. UCCACAAU 2947 0.1 9.1 1 AD- A- 3212 GUGGAGGCAGAGAA 2928- A- 3661 UUCUCUUUUCUCUGC 2926- 122318 228274 AAGAGAA 2948 2282742. CUCCACAA 2948 1.1 1.1 1 AD- A- 3213 UGGAGGCAGAGAAA 2929- A- 3662 UUUCUCUUUUCUCUG 2927- 122318 228274 AGAGAAA 2949 2282744. CCUCCACA 2949 2.1 3.1 1 AD- A- 3214 GGAGGCAGAGAAAA 2930- A- 3663 UUUUCUCUUUUCUCU 2928- 122318 228274 GAGAAAA 2950 2282746. GCCUCCAC 2950 3.1 5.1 1 AD- A- 3215 GAGGCAGAGAAAAG 2931- A- 3664 UCUUUCUCUUUUCUC 2929- 122318 228274 AGAAAGA 2951 2282748. UGCCUCCA 2951 4.1 7.1 1 AD- A- 3216 AGGCAGAGAAAAGA 2932- A- 3665 UACUUUCUCUUUUCU 2930- 122318 228274 GAAAGUA 2952 2282750. CUGCCUCC 2952 5.1 9.1 1 AD- A- 3217 GGCAGAGAAAAGAG 2933- A- 3666 UCACUUUCUCUUUUC 2931- 122318 228275 AAAGUGA 2953 2282752. UCUGCCUC 2953 6.1 1.1 1 AD- A- 3218 GCAGAGAAAAGAGA 2934- A- 3667 UACACUUUCUCUUUU 2932- 122318 228275 AAGUGUA 2954 2282754. CUCUGCCU 2954 7.1 3.1 1 AD- A- 3219 CAGAGAAAAGAGAA 2935- A- 3668 UAACACUUUCUCUUU 2933- 122318 228275 AGUGUUA 2955 2282756. UCUCUGCC 2955 8.1 5.1 1 AD- A- 3220 AGAGAAAAGAGAAA 2936- A- 3669 UAAACACUUUCUCUU 2934- 122318 228275 GUGUUUA 2956 2282758. UUCUCUGC 2956 9.1 7.1 1 AD- A- 3221 GAGAAAAGAGAAAG 2937- A- 3670 UAAAACACUUUCUCU 2935- 122319 228275 UGUUUUA 2957 2282760. UUUCUCUG 2957 0.1 9.1 1 AD- A- 3222 AGAAAAGAGAAAGU 2938- A- 3671 UUAAAACACUUUCUC 2936- 122319 228276 GUUUUAA 2958 2282762. UUUUCUCU 2958 1.1 1.1 1 AD- A- 3223 GAAAAGAGAAAGUG 2939- A- 3672 UAUAAAACACUUUCU 2937- 122319 228276 UUUUAUA 2959 2282764. CUUUUCUC 2959 2.1 3.1 1 AD- A- 3224 AAAAGAGAAAGUGU 2940- A- 3673 UUAUAAAACACUUUC 2938- 122319 228276 UUUAUAA 2960 2282766. UCUUUUCU 2960 3.1 5.1 1 AD- A- 3225 AAAGAGAAAGUGUU 2941- A- 3674 UAUAUAAAACACUUU 2939- 122319 228276 UUAUAUA 2961 2282768. CUCUUUUC 2961 4.1 7.1 1 AD- A- 3226 AAGAGAAAGUGUUU 2942- A- 3675 UUAUAUAAAACACUU 2940- 122319 228276 UAUAUAA 2962 2282770. UCUCUUUU 2962 5.1 9.1 1 AD- A- 3227 AGAGAAAGUGUUUU 2943- A- 3676 UGUAUAUAAAACACU 2941- 122319 228277 AUAUACA 2963 2282772. UUCUCUUU 2963 6.1 1.1 1 AD- A- 3228 GAGAAAGUGUUUUA 2944- A- 3677 UCGUAUAUAAAACAC 2942- 122319 228277 UAUACGA 2964 2282774. UUUCUCUU 2964 7.1 3.1 1 AD- A- 3229 AGAAAGUGUUUUAU 2945- A- 3678 UCCGUAUAUAAAACA 2943- 122319 228277 AUACGGA 2965 2282776. CUUUCUCU 2965 8.1 5.1 1 AD- A- 3230 AAAGUGUUUUAUAU 2947- A- 3679 UUACCGUAUAUAAAA 2945- 122319 228277 ACGGUAA 2967 2282778. CACUUUCU 2967 9.1 7.1 1 AD- A- 3231 AAGUGUUUUAUAUA 2948- A- 3680 UGUACCGUAUAUAAA 2946- 122320 228277 CGGUACA 2968 2282780. ACACUUUC 2968 0.1 9.1 1 AD- A- 3232 AGUGUUUUAUAUAC 2949- A- 3681 UAGUACCGUAUAUAA 2947- 122320 228278 GGUACUA 2969 2282782. AACACUUU 2969 1.1 1.1 1 AD- A- 3233 GUGUUUUAUAUACG 2950- A- 3682 UAAGUACCGUAUAUA 2948- 122320 228278 GUACUUA 2970 2282784. AAACACUU 2970 2.1 3.1 1 AD- A- 3234 UGUUUUAUAUACGG 2951- A- 3683 UUAAGUACCGUAUAU 2949- 122320 228278 UACUUAA 2971 2282786. AAAACACU 2971 3.1 5.1 1 AD- A- 3235 GUUUUAUAUACGGU 2952- A- 3684 UAUAAGUACCGUAUA 2950- 122320 228278 ACUUAUA 2972 2282788. UAAAACAC 2972 4.1 7.1 1 AD- A- 3236 UUUUAUAUACGGUA 2953- A- 3685 UAAUAAGUACCGUAU 2951- 122320 228278 CUUAUUA 2973 2282790. AUAAAACA 2973 5.1 9.1 1 AD- A- 3237 UUUAUAUACGGUAC 2954- A- 3686 UAAAUAAGUACCGUA 2952- 122320 228279 UUAUUUA 2974 2282792. UAUAAAAC 2974 6.1 1.1 1 AD- A- 3238 UUAUAUACGGUACU 2955- A- 3687 UUAAAUAAGUACCGU 2953- 122320 228279 UAUUUAA 2975 2282794. AUAUAAAA 2975 7.1 3.1 1 AD- A- 3239 UAUAUACGGUACUU 2956- A- 3688 UUUAAAUAAGUACCG 2954- 122320 228279 AUUUAAA 2976 2282796. UAUAUAAA 2976 8.1 5.1 1 AD- A- 3240 GUACUUAUUUAAUA 2964- A- 3689 UAAGGGAUAUUAAAU 2962- 122320 228279 UCCCUUA 2984 2282798. AAGUACCG 2984 9.1 7.1 1 AD- A- 3241 UACUUAUUUAAUAU 2965- A- 3690 UAAAGGGAUAUUAAA 2963- 122321 228279 CCCUUUA 2985 2282800. UAAGUACC 2985 0.1 9.1 1 AD- A- 3242 UUAUGAGAUGUAUC 3068- A- 3691 UGCAAAAGAUACAUC 3066- 122321 228280 UUUUGCA 3088 2282802. UCAUAAAU 3088 1.1 1.1 1 AD- A- 3243 UAUGAGAUGUAUCU 3069- A- 3692 UAGCAAAAGAUACAU 3067- 122321 228280 UUUGCUA 3089 2282804. CUCAUAAA 3089 2.1 3.1 1 AD- A- 3244 AUGAGAUGUAUCUU 3070- A- 3693 UGAGCAAAAGAUACA 3068- 122321 228280 UUGCUCA 3090 2282806. UCUCAUAA 3090 3.1 5.1 1 AD- A- 3245 UGAGAUGUAUCUUU 3071- A- 3694 UAGAGCAAAAGAUAC 3069- 122321 228280 UGCUCUA 3091 2282808. AUCUCAUA 3091 4.1 7.1 1 AD- A- 3246 GAGAUGUAUCUUUU 3072- A- 3695 UGAGAGCAAAAGAUA 3070- 122321 228280 GCUCUCA 3092 2282810. CAUCUCAU 3092 5.1 9.1 1 AD- A- 3247 AGAUGUAUCUUUUG 3073- A- 3696 UAGAGAGCAAAAGAU 3071- 122321 228281 CUCUCUA 3093 2282812. ACAUCUCA 3093 6.1 1.1 1 AD- A- 3248 GAUGUAUCUUUUGC 3074- A- 3697 UGAGAGAGCAAAAGA 3072- 122321 228281 UCUCUCA 3094 2282814. UACAUCUC 3094 7.1 3.1 1 AD- A- 3249 AUGUAUCUUUUGCUC 3075- A- 3698 UAGAGAGAGCAAAAG 3073- 122321 228281 UCUCUA 3095 2282816. AUACAUCU 3095 8.1 5.1 1 AD- A- 3250 GCUCUCUCUUGCUCU 3086- A- 3699 UAUAAGAGAGCAAGA 3084- 122321 228281 CUUAUA 3106 2282818. GAGAGCAA 3106 9.1 7.1 1 AD- A- 3251 CUCUCUCUUGCUCUC 3087- A- 3700 UAAUAAGAGAGCAAG 3085- 122322 228281 UUAUUA 3107 2282820. AGAGAGCA 3107 0.1 9.1 1 AD- A- 3252 UCUCUCUUGCUCUCU 3088- A- 3701 UAAAUAAGAGAGCAA 3086- 122322 228282 UAUUUA 3108 2282822. GAGAGAGC 3108 1.1 1.1 1 AD- A- 3253 CUCUCUUGCUCUCUU 3089- A- 3702 UCAAAUAAGAGAGCA 3087- 122322 228282 AUUUGA 3109 2282824. AGAGAGAG 3109 2.1 3.1 1 AD- A- 3254 UCUCUUGCUCUCUUA 3090- A- 3703 UACAAAUAAGAGAGC 3088- 122322 228282 UUUGUA 3110 2282826. AAGAGAGA 3110 3.1 5.1 1 AD- A- 3255 CUCUUGCUCUCUUAU 3091- A- 3704 UUACAAAUAAGAGAG 3089- 122322 228282 UUGUAA 3111 2282828. CAAGAGAG 3111 4.1 7.1 1 AD- A- 3256 UCUUGCUCUCUUAUU 3092- A- 3705 UGUACAAAUAAGAGA 3090- 122322 228282 UGUACA 3112 2282830. GCAAGAGA 3112 5.1 9.1 1 AD- A- 3257 CUUGCUCUCUUAUUU 3093- A- 3706 UGGUACAAAUAAGAG 3091- 122322 228283 GUACCA 3113 2282832. AGCAAGAG 3113 6.1 1.1 1 AD- A- 3258 UUGCUCUCUUAUUUG 3094- A- 3707 UCGGUACAAAUAAGA 3092- 122322 228283 UACCGA 3114 2282834. GAGCAAGA 3114 7.1 3.1 1 AD- A- 3259 UGCUCUCUUAUUUGU 3095- A- 3708 UCCGGUACAAAUAAG 3093- 122322 228283 ACCGGA 3115 2282836. AGAGCAAG 3115 8.1 5.1 1 AD- A- 3260 CUCUCUUAUUUGUAC 3097- A- 3709 UAACCGGUACAAAUA 3095- 122322 228283 CGGUUA 3117 2282838. AGAGAGCA 3117 9.1 7.1 1 AD- A- 3261 UCUCUUAUUUGUACC 3098- A- 3710 UAAACCGGUACAAAU 3096- 122323 228283 GGUUUA 3118 2282840. AAGAGAGC 3118 0.1 9.1 1 AD- A- 3262 CUCUUAUUUGUACCG 3099- A- 3711 UAAAACCGGUACAAA 3097- 122323 228284 GUUUUA 3119 2282842. UAAGAGAG 3119 1.1 1.1 1 AD- A- 3263 UCUUAUUUGUACCGG 3100- A- 3712 UAAAAACCGGUACAA 3098- 122323 228284 UUUUUA 3120 2282844. AUAAGAGA 3120 2.1 3.1 1 AD- A- 3264 CUUAUUUGUACCGGU 3101- A- 3713 UCAAAAACCGGUACA 3099- 122323 228284 UUUUGA 3121 2282846. AAUAAGAG 3121 3.1 5.1 1 AD- A- 3265 UUAUUUGUACCGGU 3102- A- 3714 UACAAAAACCGGUAC 3100- 122323 228284 UUUUGUA 3122 2282848. AAAUAAGA 3122 4.1 7.1 1 AD- A- 3266 UAUUUGUACCGGUU 3103- A- 3715 UUACAAAAACCGGUA 3101- 122323 228284 UUUGUAA 3123 2282850. CAAAUAAG 3123 5.1 9.1 1 AD- A- 3267 AUUUGUACCGGUUU 3104- A- 3716 UAUACAAAAACCGGU 3102- 122323 228285 UUGUAUA 3124 2282852. ACAAAUAA 3124 6.1 1.1 1 AD- A- 3268 UUUGUACCGGUUUU 3105- A- 3717 UUAUACAAAAACCGG 3103- 122323 228285 UGUAUAA 3125 2282854. UACAAAUA 3125 7.1 3.1 1 AD- A- 3269 UUGUACCGGUUUUU 3106- A- 3718 UAUAUACAAAAACCG 3104- 122323 228285 GUAUAUA 3126 2282856. GUACAAAU 3126 8.1 5.1 1 AD- A- 3270 UGUACCGGUUUUUG 3107- A- 3719 UUAUAUACAAAAACC 3105- 122323 228285 UAUAUAA 3127 2282858. GGUACAAA 3127 9.1 7.1 1 AD- A- 3271 GUACCGGUUUUUGU 3108- A- 3720 UUUAUAUACAAAAAC 3106- 122324 228285 AUAUAAA 3128 2282860. CGGUACAA 3128 0.1 9.1 1 AD- A- 3272 UACCGGUUUUUGUA 3109- A- 3721 UUUUAUAUACAAAAA 3107- 122324 228286 UAUAAAA 3129 2282862. CCGGUACA 3129 1.1 1.1 1 AD- A- 3273 ACCGGUUUUUGUAU 3110- A- 3722 UUUUUAUAUACAAAA 3108- 122324 228286 AUAAAAA 3130 2282864. ACCGGUAC 3130 2.1 3.1 1 AD- A- 3274 AUUCAUGUUUCCAAU 3129- A- 3723 UGAGAGAUUGGAAAC 3127- 122324 228286 CUCUCA 3149 2282866. AUGAAUUU 3149 3.1 5.1 1 AD- A- 3275 UUCAUGUUUCCAAUC 3130- A- 3724 UAGAGAGAUUGGAAA 3128- 122324 228286 UCUCUA 3150 2282868. CAUGAAUU 3150 4.1 7.1 1 AD- A- 3276 UCAUGUUUCCAAUCU 3131- A- 3725 UGAGAGAGAUUGGAA 3129- 122324 228286 CUCUCA 3151 2282870. ACAUGAAU 3151 5.1 9.1 1 AD- A- 3277 CAUGUUUCCAAUCUC 3132- A- 3726 UAGAGAGAGAUUGGA 3130- 122324 228287 UCUCUA 3152 2282872. AACAUGAA 3152 6.1 1.1 1 AD- A- 3278 AUGUUUCCAAUCUCU 3133- A- 3727 UGAGAGAGAGAUUGG 3131- 122324 228287 CUCUCA 3153 2282874. AAACAUGA 3153 7.1 3.1 1 AD- A- 3279 UGUUUCCAAUCUCUC 3134- A- 3728 UGGAGAGAGAGAUUG 3132- 122324 228287 UCUCCA 3154 2282876. GAAACAUG 3154 8.1 5.1 1 AD- A- 3280 UUUCCAAUCUCUCUC 3136- A- 3729 UAGGGAGAGAGAGAU 3134- 122324 228287 UCCCUA 3156 2282878. UGGAAACA 3156 9.1 7.1 1 AD- A- 3281 UCCAAUCUCUCUCUC 3138- A- 3730 UUCAGGGAGAGAGAG 3136- 122325 228287 CCUGAA 3158 2282880. AUUGGAAA 3158 0.1 9.1 1 AD- A- 3282 CGGUGACAGUCACUA 3159- A- 3731 UUAAGCUAGUGACUG 3157- 122325 228288 GCUUAA 3179 2282882. UCACCGAU 3179 1.1 1.1 1 AD- A- 3283 GGUGACAGUCACUAG 3160- A- 3732 UAUAAGCUAGUGACU 3158- 122325 228288 CUUAUA 3180 2282884. GUCACCGA 3180 2.1 3.1 1 AD- A- 3284 AGUCACUAGCUUAUC 3166- A- 3733 UUUCAAGAUAAGCUA 3164- 122325 228288 UUGAAA 3186 2282886. GUGACUGU 3186 3.1 5.1 1 AD- A- 3285 GUCACUAGCUUAUCU 3167- A- 3734 UGUUCAAGAUAAGCU 3165- 122325 228288 UGAACA 3187 2282888. AGUGACUG 3187 4.1 7.1 1 AD- A- 3286 UCACUAGCUUAUCUU 3168- A- 3735 UUGUUCAAGAUAAGC 3166- 122325 228288 GAACAA 3188 2282890. UAGUGACU 3188 5.1 9.1 1 AD- A- 3287 ACUAGCUUAUCUUGA 3170- A- 3736 UUCUGUUCAAGAUAA 3168- 122325 228289 ACAGAA 3190 2282892. GCUAGUGA 3190 6.1 1.1 1 AD- A- 3288 CUAGCUUAUCUUGAA 3171- A- 3737 UAUCUGUUCAAGAUA 3169- 122325 228289 CAGAUA 3191 2282894. AGCUAGUG 3191 7.1 3.1 1 AD- A- 3289 UAGCUUAUCUUGAAC 3172- A- 3738 UUAUCUGUUCAAGAU 3 no- 122325 228289 AGAUAA 3192 2282896. AAGCUAGU 3192 8.1 5.1 1 AD- A- 3290 AGCUUAUCUUGAACA 3173- A- 3739 UAUAUCUGUUCAAGA 3171- 122325 228289 GAUAUA 3193 2282898. UAAGCUAG 3193 9.1 7.1 1 AD- A- 3291 GCUUAUCUUGAACAG 3174- A- 3740 UAAUAUCUGUUCAAG 3172- 122326 228289 AUAUUA 3194 2282900. AUAAGCUA 3194 0.1 9.1 1 AD- A- 3292 CAGCACACAUUCCUU 3241- A- 3741 UUUUCAAAGGAAUGU 3239- 122326 228290 UGAAAA 3261 2282902. GUGCUGGG 3261 1.1 1.1 1 AD- A- 3293 AGCACACAUUCCUUU 3242- A- 3742 UAUUUCAAAGGAAUG 3240- 122326 228290 GAAAUA 3262 2282904. UGUGCUGG 3262 2.1 3.1 1 AD- A- 3294 GCACACAUUCCUUUG 3243- A- 3743 UUAUUUCAAAGGAAU 3241- 122326 228290 AAAUAA 3263 2282906. GUGUGCUG 3263 3.1 5.1 1 AD- A- 3295 CACACAUUCCUUUGA 3244- A- 3744 UUUAUUUCAAAGGAA 3242- 122326 228290 AAUAAA 3264 2282908. UGUGUGCU 3264 4.1 7.1 1 AD- A- 3296 CACAUUCCUUUGAAA 3246- A- 3745 UCCUUAUUUCAAAGG 3244- 122326 228290 UAAGGA 3266 2282910. AAUGUGUG 3266 5.1 9.1 1 AD- A- 3297 UCCUUUGAAAUAAG 3251- A- 3746 UUGAAACCUUAUUUC 3249- 122326 228291 GUUUCAA 3271 2282912. AAAGGAAU 3271 6.1 1.1 1 AD- A- 3298 CUUUGAAAUAAGGU 3253- A- 3747 UAUUGAAACCUUAUU 3251- 122326 228291 UUCAAUA 3273 2282914. UCAAAGGA 3273 7.1 3.1 1 AD- A- 3299 UUUGAAAUAAGGUU 3254- A- 3748 UUAUUGAAACCUUAU 3252- 122326 228291 UCAAUAA 3274 2282916. UUCAAAGG 3274 8.1 5.1 1 AD- A- 3300 AGGUUUCAAUAUAC 3263- A- 3749 UGUAGAUGUAUAUUG 3261- 122326 228291 AUCUACA 3283 2282918. AAACCUUA 3283 9.1 7.1 1 AD- A- 3301 GGUUUCAAUAUACA 3264- A- 3750 UUGUAGAUGUAUAUU 3262- 122327 228291 UCUACAA 3284 2282920. GAAACCUU 3284 0.1 9.1 1 AD- A- 3302 GUUUCAAUAUACAUC 3265- A- 3751 UAUGUAGAUGUAUAU 3263- 122327 228292 UACAUA 3285 2282922. UGAAACCU 3285 1.1 1.1 1 AD- A- 3303 UUUCAAUAUACAUCU 3266- A- 3752 UUAUGUAGAUGUAUA 3264- 122327 228292 ACAUAA 3286 2282924. UUGAAACC 3286 2.1 3.1 1 AD- A- 3304 UUCAAUAUACAUCUA 3267- A- 3753 UGUAUGUAGAUGUAU 3265- 122327 228292 CAUACA 3287 2282926. AUUGAAAC 3287 3.1 5.1 1 AD- A- 3305 UAUUUGGCAACUUG 3295- A- 3754 UCAAAUACAAGUUGC 3293- 122327 228292 UAUUUGA 3315 2282928. CAAAUAUA 3315 4.1 7.1 1 AD- A- 3306 UUUGGCAACUUGUA 3297- A- 3755 UCACAAAUACAAGUU 3295- 122327 228292 UUUGUGA 3317 2282930. GCCAAAUA 3317 5.1 9.1 1 AD- A- 3307 UGGCAACUUGUAUU 3299- A- 3756 UCACACAAAUACAAG 3297- 122327 228293 UGUGUGA 3319 2282932. UUGCCAAA 3319 6.1 1.1 1 AD- A- 3308 GGCAACUUGUAUUU 3300- A- 3757 UACACACAAAUACAA 3298- 122327 228293 GUGUGUA 3320 2282934. GUUGCCAA 3320 7.1 3.1 1 AD- A- 3309 GCAACUUGUAUUUG 3301- A- 3758 UUACACACAAAUACA 3299- 122327 228293 UGUGUAA 3321 2282936. AGUUGCCA 3321 8.1 5.1 1 AD- A- 3310 CAACUUGUAUUUGU 3302- A- 3759 UAUACACACAAAUAC 3300- 122327 228293 GUGUAUA 3322 2282938. AAGUUGCC 3322 9.1 7.1 1 AD- A- 3311 AACUUGUAUUUGUG 3303- A- 3760 UUAUACACACAAAUA 3301- 122328 228293 UGUAUAA 3323 2282940. CAAGUUGC 3323 0.1 9.1 1 AD- A- 3312 ACUUGUAUUUGUGU 3304- A- 3761 UAUAUACACACAAAU 3302- 122328 228294 GUAUAUA 3324 2282942. ACAAGUUG 3324 1.1 1.1 1 AD- A- 3313 UUCUGAUAAAAUAG 3354- A- 3762 UCAAUGUCUAUUUUA 3352- 122328 228294 ACAUUGA 3374 2282944. UCAGAAUC 3374 2.1 3.1 1 AD- A- 3314 UGAUAAAAUAGACA 3357- A- 3763 UUAGCAAUGUCUAUU 3355- 122328 228294 UUGCUAA 3377 2282946. UUAUCAGA 3377 3.1 5.1 1 AD- A- 3315 GAUAAAAUAGACAU 3358- A- 3764 UAUAGCAAUGUCUAU 3356- 122328 228294 UGCUAUA 3378 2282948. UUUAUCAG 3378 4.1 7.1 1 AD- A- 3316 UAAAAUAGACAUUG 3360- A- 3765 UGAAUAGCAAUGUCU 3358- 122328 228294 CUAUUCA 3380 2282950. AUUUUAUC 3380 5.1 9.1 1 AD- A- 3317 UAGACAUUGCUAUUC 3365- A- 3766 UAAACAGAAUAGCAA 3363- 122328 228295 UGUUUA 3385 2282952. UGUCUAUU 3385 6.1 1.1 1 AD- A- 3318 AGACAUUGCUAUUCU 3366- A- 3767 UAAAACAGAAUAGCA 3364- 122328 228295 GUUUUA 3386 2282954. AUGUCUAU 3386 7.1 3.1 1 AD- A- 3319 UCUACAUACUAAAUC 3422- A- 3768 UAGAGAGAUUUAGUA 3420- 122328 228295 UCUCUA 3442 2282956. UGUAGAAU 3442 8.1 5.1 1 AD- A- 3320 CUACAUACUAAAUCU 3423- A- 3769 UGAGAGAGAUUUAGU 3421- 122328 228295 CUCUCA 3443 2282958. AUGUAGAA 3443 9.1 7.1 1 AD- A- 3321 UACAUACUAAAUCUC 3424- A- 3770 UGGAGAGAGAUUUAG 3422- 122329 228295 UCUCCA 3444 2282960. UAUGUAGA 3444 0.1 9.1 1 AD- A- 3322 ACAUACUAAAUCUCU 3425- A- 3771 UAGGAGAGAGAUUUA 3423- 122329 228296 CUCCUA 3445 2282962. GUAUGUAG 3445 1.1 1.1 1 AD- A- 3323 CAUACUAAAUCUCUC 3426- A- 3772 UAAGGAGAGAGAUUU 3424- 122329 228296 UCCUUA 3446 2282964. AGUAUGUA 3446 2.1 3.1 1 AD- A- 3324 AUACUAAAUCUCUCU 3427- A- 3773 UAAAGGAGAGAGAUU 3425- 122329 228296 CCUUUA 3447 2282966. UAGUAUGU 3447 3.1 5.1 1 AD- A- 3325 UACUAAAUCUCUCUC 3428- A- 3774 UAAAAGGAGAGAGAU 3426- 122329 228296 CUUUUA 3448 2282968. UUAGUAUG 3448 4.1 7.1 1 AD- A- 3326 CAUUUAUUUAUUGG 3468- A- 3775 UGUAGCACCAAUAAA 3466- 122329 228296 UGCUACA 3488 2282970. UAAAUGAU 3488 5.1 9.1 1 AD- A- 3327 AUUUAUUUAUUGGU 3469- A- 3776 UAGUAGCACCAAUAA 3467- 122329 228297 GCUACUA 3489 2282972. AUAAAUGA 3489 6.1 1.1 1 AD- A- 3328 UUUAUUUAUUGGUG 3470- A- 3777 UCAGUAGCACCAAUA 3468- 122329 228297 CUACUGA 3490 2282974. AAUAAAUG 3490 7.1 3.1 1 AD- A- 3329 UUAUUUAUUGGUGC 3471- A- 3778 UACAGUAGCACCAAU 3469- 122329 228297 UACUGUA 3491 2282976. AAAUAAAU 3491 8.1 5.1 1 AD- A- 3330 UAUUUAUUGGUGCU 3472- A- 3779 UAACAGUAGCACCAA 3470- 122329 228297 ACUGUUA 3492 2282978. UAAAUAAA 3492 9.1 7.1 1 AD- A- 3331 AUUUAUUGGUGCUA 3473- A- 3780 UAAACAGUAGCACCA 3471- 122330 228297 CUGUUUA 3493 2282980. AUAAAUAA 3493 0.1 9.1 1 AD- A- 3332 UUAUUGGUGCUACU 3475- A- 3781 UAUAAACAGUAGCAC 3473- 122330 228298 GUUUAUA 3495 2282982. CAAUAAAU 3495 1.1 1.1 1 AD- A- 3333 AUUGGUGCUACUGU 3477- A- 3782 UGGAUAAACAGUAGC 3475- 122330 228298 UUAUCCA 3497 2282984. ACCAAUAA 3497 2.1 3.1 1 AD- A- 3334 GAAAAGAUAUUAAC 3511- A- 3783 UCGUGAUGUUAAUAU 3509- 122330 228298 AUCACGA 3531 2282986. CUUUUCCC 3531 3.1 5.1 1 AD- A- 3335 AACAUCACGUCUUUG 3522- A- 3784 UAGAGACAAAGACGU 3520- 122330 228298 UCUCUA 3542 2282988. GAUGUUAA 3542 4.1 7.1 1 AD- A- 3336 ACAUCACGUCUUUGU 3523- A- 3785 UUAGAGACAAAGACG 3521- 122330 228298 CUCUAA 3543 2282990. UGAUGUUA 3543 5.1 9.1 1 AD- A- 3337 GUCUUUGUCUCUAGU 3530- A- 3786 UACUGCACUAGAGAC 3528- 122330 228299 GCAGUA 3550 2282992. AAAGACGU 3550 6.1 1.1 1 AD- A- 3338 UCUUUGUCUCUAGUG 3531- A- 3787 UAACUGCACUAGAGA 3529- 122330 228299 CAGUUA 3551 2282994. CAAAGACG 3551 7.1 3.1 1 AD- A- 3339 CUUUGUCUCUAGUGC 3532- A- 3788 UAAACUGCACUAGAG 3530- 122330 228299 AGUUUA 3552 2282996. ACAAAGAC 3552 8.1 5.1 1 AD- A- 3340 GAGAUAUUCCGUAG 3555- A- 3789 UUAUGUACUACGGAA 3553- 122330 228299 UACAUAA 3575 2282998. UAUCUCGA 3575 9.1 7.1 1 AD- A- 3341 AGAUAUUCCGUAGU 3556- A- 3790 UAUAUGUACUACGGA 3554- 122331 228299 ACAUAUA 3576 2283000. AUAUCUCG 3576 0.1 9.1 1 AD- A- 3342 GAUAUUCCGUAGUAC 3557- A- 3791 UAAUAUGUACUACGG 3555- 122331 228300 AUAUUA 3577 2283002. AAUAUCUC 3577 1.1 1.1 1 AD- A- 3343 CGACAAAGAAAUACA 3590- A- 3792 UAUAUCUGUAUUUCU 3588- 122331 228300 GAUAUA 3610 2283004. UUGUCGUU 3610 2.1 3.1 1 AD- A- 3344 GACAAAGAAAUACA 3591- A- 3793 UUAUAUCUGUAUUUC 3589- 122331 228300 GAUAUAA 3611 2283006. UUUGUCGU 3611 3.1 5.1 1 AD- A- 3345 ACAAAGAAAUACAG 3592- A- 3794 UAUAUAUCUGUAUUU 3590- 122331 228300 AUAUAUA 3612 2283008. CUUUGUCG 3612 4.1 7.1 1 AD- A- 3346 CAAAGAAAUACAGA 3593- A- 3795 UGAUAUAUCUGUAUU 3591- 122331 228300 UAUAUCA 3613 2283010. UCUUUGUC 3613 5.1 9.1 1

TABLE 10A Exemplary Human VEGF-A siRNA Modified Single Strands and Duplex Sequences Anti- SEQ ID mRNA Target SEQ ID Sense SEQ sense NO: Sequence NO: Duplex Oligo ID NO: Oligo (Anti- (mRNA Name Name (Sense) Sense Sequence Name sense) Antisense Sequence target) AD- A- 3796 asasaag(Ahd)gadAadGugu A- 3886 VPusdTsaudAadAaca AGAAAAGAGAAA 5149 1353514.1 2521322.1 uuuausasa 2521323.1 cdTudTcdTcuuuuscsu GUGUUUUAUAU AD- A- 3797 asasaag(Ahd)gaAfAfGfug A- 3887 VPusdTsaudAadAaca AGAAAAGAGAAA 5150 1353484.1 2521264.1 uuuuausasa 2521265.1 cdTuUfcucuuuuscsu GUGUUUUAUAU AD- A- 3798 asasaag(Ahd)GfaAfAfGfu A- 3888 VPusUfsauaAfaacacu AGAAAAGAGAAA 5151 1353454.1 2521212.1 guuuuausasa 2282766.1 uUfcUfcuuuuscsu GUGUUUUAUAU AD- A- 3799 asasaga(Chd)ugAfUfAfca A- 3889 VPusdAsucdGudTcug GGAAAGACUGAU 5152 1353468.1 2521232.1 gaacgasusa 2521233.1 udAuCfagucuuuscsc ACAGAACGAUC AD- A- 3800 asasaga(Chd)ugdAudAcag A- 3890 VPusdAsucdGudTcug GGAAAGACUGAU 5153 1353498.1 2521292.1 aacgasusa 1800369.1 udAudCadGucuuuscsc ACAGAACGAUC AD- A- 3801 asasaga(Chd)UfgAfUfAfc A- 3891 VPusAfsucgUfucugua GGAAAGACUGAU 5154 1353438.1 1700819.1 agaacgasusa 2282346.1 uCfaGfucuuuscsc ACAGAACGAUC AD- A- 3802 asasagagaadAgdTguuu A- 3892 VPusdAsuadTadAaac GAAAAGAGAAAG 5155 1353515.1 2521324.1 (Uhd)auasusa 2521325.1 adCudTudCucuuususc UGUUUUAUAUA AD- A- 3803 asasagagaaAfGfUfguuu A- 3893 VPusdAsuadTadAaac GAAAAGAGAAAG 5156 1353485.1 2521266.1 (Uhd)auasusa 2521267.1 adCuUfucucuuususc UGUUUUAUAUA AD- A- 3804 asasagagAfaAfGfUfguuu A- 3894 VPusAfsuauAfaaacac GAAAAGAGAAAG 5157 1353455.1 2521213.1 (Uhd)auasusa 2282768.1 uUfuCfucuuususc UGUUUUAUAUA AD- A- 3805 asascag(Uhd)gcdTadAugu A- 3895 VPusdCscadAudAaca UUAACAGUGCUA 5158 1353513.1 2521320.1 uauugsgsa 2521321.1 udTadGcdAcuguusgsg AUGUUAUUGGU AD- A- 3806 asascag(Uhd)gcUfAfAfug A- 3896 VPusdCscadAudAaca UUAACAGUGCUA 5159 1353483.1 2521262.1 uuauugsgsa 2521263.1 udTaGfcacuguusgsg AUGUUAUUGGU AD- A- 3807 asascag(Uhd)GfcUfAfAfu A- 3897 VPusCfscaaUfaacauua UUAACAGUGCUA 5160 1353453.1 1700832.1 guuauugsgsa 2521211.1 GfcAfcuguusgsg AUGUUAUUGGU AD- A- 3808 asascga(Uhd)cgdAudAcag A- 3898 VPusdTsggdTu(U2p)c AGAACGAUCGAU 5161 1353502.1 2521298.1 aaaccsasa 2521299.1 ugudAudCgdAucguus ACAGAAACCAC AD- A- 3809 asascga(Uhd)cgAfUfAfca A- 3899 VPusdTsggdTu(U2p)c AGAACGAUCGAU 5162 1353472.1 2521240.1 gaaaccsasa 2521241.1 ugudAuCfgaucguuscs ACAGAAACCAC AD- A- 3810 asascga(Uhd)CfgAfUfAfc A- 3900 VPusUfsggdTu(U2p)c AGAACGAUCGAU 5163 1353442.1 2521195.1 agaaaccsasa 2521196.1 uguauCfgAfucguuscsu ACAGAAACCAC AD- A- 3811 asasgac(Uhd)gadTadCaga A- 3901 VPusdGsaudCg(U2p) GAAAGACUGAUA 5164 1353499.1 2483623.1 acgauscsa 2521293.1 ucugdTadTedAgucuus CAGAACGAUCG AD- A- 3812 asasgac(Uhd)gaUfAfCfag A- 3902 VPusdGsaudCg(U2p) GAAAGACUGAUA 5165 1353469.1 2521234.1 aacgauscsa 2521235.1 ucugdTaUfcagucuusus CAGAACGAUCG AD- A- 3813 asasgac(Uhd)GfaUfAfCfa A- 3903 VPusGfsaudCg(U2p)u GAAAGACUGAUA 5166 1353439.1 1700820.1 gaacgauscsa 2521191.1 cuguaUfcAfgucuususc CAGAACGAUCG AD- A- 3814 asasgag(Ahd)aadGudGuu A- 3904 VPusdTsaudAudAaaa AAAAGAGAAAGU 5167 1353516.1 2521326.1 uuauausasa 2521327.1 cdAcdTudTcucuususu GUUUUAUAUAC AD- A- 3815 asasgag(Ahd)aaGfUfGfuu A- 3905 VPusdTsaudAudAaaa AAAAGAGAAAGU 5168 1353486.1 2521268.1 uuauausasa 2521269.1 cdAcUfuucucuususu GUUUUAUAUAC AD- A- 3816 asasgag(Ahd)AfaGfUfGfu A- 3906 VPusUfsauaUfaaaacac AAAAGAGAAAGU 5169 1353456.1 2521214.1 uuuauausasa 2282770.1 UfuUfcucuususu GUUUUAUAUAC AD- A- 3817 asasuuggaudTcdGccau A- 3907 VPusdAsuadAadAugg GGAAUUGGAUUC 5170 1353509.1 2521312.1 (Uhd)uuasusa 2521313.1 cdGadAudCcaauuscsc GCCAUUUUAUU AD- A- 3818 asasuuggauUfCfGfccau A- 3908 VPusdAsuadAadAugg GGAAUUGGAUUC 5171 1353479.1 2521254.1 (Uhd)uuasusa 2521255.1 cdGaAfuccaauuscsc GCCAUUUUAUU AD- A- 3819 asasuuggAfuUfCfGfccau A- 3909 VPusAfsuaaAfauggcg GGAAUUGGAUUC 5172 1353449.1 2521206.1 (Uhd)uuasusa 2282454.1 aAfuCfcaauuscsc GCCAUUUUAUU AD- A- 3820 ascsaga(Ahd)cadGudCcuu A- 3910 VPusdTsggdAudTaag CGACAGAACAGUC 5173 1353503.1 2521300.1 aauccsasa 2521301.1 gdAcdTgdTucuguscsg CUUAAUCCAG AD- A- 3821 ascsaga(Ahd)caGfUfCfcu A- 3911 VPusdTsggdAudTaag CGACAGAACAGUC 5174 1353473.1 2521242.1 uaauccsasa 2521243.1 gdAcUfguucuguscsg CUUAAUCCAG AD- A- 3822 ascsaga(Ahd)CfaGfUfCfc A- 3912 VPusUfsggaUfuaagga CGACAGAACAGUC 5175 1353443.1 2521197.1 uuaauccsasa 2282402.1 cUfgUfucuguscsg CUUAAUCCAG AD- A- 3823 ascsagu(Chd)cudTadAucc A- 3913 VPusdGsuudTc(U2p)g GAACAGUCCUUAA 5176 1353506.1 2521306.1 agaaascsa 2521307.1 gaudTadAgdGacugusu UCCAGAAACC AD- A- 3824 ascsagu(Chd)cuUfAfAfuc A- 3914 VPusdGsuudTc(U2p)g GAACAGUCCUUAA 5177 1353476.1 2521248.1 cagaaascsa 2521249.1 gaudTaAfggacugususc UCCAGAAACC AD- A- 3825 ascsagu(Chd)CfuUfAfAfu A- 3915 VPusGfsuudTc(U2p)g GAACAGUCCUUAA 5178 1353446.1 2521201.1 ccagaaascsa 2521202.1 gauuaAfgGfacugususc UCCAGAAACC AD- A- 3826 ascscaggaadAgdAcuga A- 3916 VPusdCsugdTadTcag UCACCAGGAAAGA 5179 1353497.1 2521290.1 (Uhd)acasgsa 2521291.1 udCudTudCcuggusgsc CUGAUACAGA AD- A- 3827 ascscaggaaAfGfAfcuga A- 3917 VPusdCsugdTadTcag UCACCAGGAAAGA 5180 1353467.1 2521230.1 (Uhd)acasgsa 2521231.1 udCuUfuccuggusgsc CUGAUACAGA AD- A- 3828 ascscaggAfaAfGfAfcuga A- 3918 VPusCfsuguAfucaguc UCACCAGGAAAGA 5181 1353437.1 2521189.1 (Uhd)acasgsa 2521190.1 uUfuCfcuggusgsc CUGAUACAGA AD- A- 3829 ascscaugcadGadTuaug A- 3919 VPusdAsucdCg(C2p)a UCACCAUGCAGAU 5182 1353494.1 2521284.1 (Chd)ggasusa 2521285.1 uaadTcdTgdCauggusg UAUGCGGAUC AD- A- 3830 ascscaugcaGfAfUfuaug A- 3920 VPusdAsucdCg(C2p)a UCACCAUGCAGAU 5183 1353464.1 2521224.1 (Chd)ggasusa 2521225.1 uaadTcUfgcauggusgsc UAUGCGGAUC AD- A- 3831 ascscaugCfaGfAfUfuaug A- 3921 VPusAfsucdCg(C2p)a UCACCAUGCAGAU 5184 1353434.1 2521183.1 (Chd)ggasusa 2521184.1 uaaucUfgCfauggusgsc UAUGCGGAUC AD- A- 3832 asgsaac(Ahd)gudCcdTuaa A- 3922 VPusdTscudGg(A2p)u ACAGAACAGUCCU 5185 1353505.1 2521304.1 uccagsasa 2521305.1 uaadGgdAcdT guucusg UAAUCCAGAA AD- A- 3833 asgsaac(Ahd)guCfCfUfua A- 3923 VPusdTscudGg(A2p)u ACAGAACAGUCCU 5186 1353475.1 2521246.1 auccagsasa 2521247.1 uaadGgAfcuguucusgs UAAUCCAGAA AD- A- 3834 asgsaac(Ahd)GfuCfCfUfu A- 3924 VPusUfscudGg(A2p)u ACAGAACAGUCCU 5187 1353445.1 2521199.1 aauccagsasa 2521200.1 uaaggAfcUfguucusgsu UAAUCCAGAA AD- A- 3835 asgsaug(Uhd)audCudTuug A- 3925 VPusdAsgadGa(G2p)c UGAGAUGUAUCU 5188 1353518.1 2521330.1 cucucsusa 2521331.1 aaadAgdAudAcaucusc UUUGCUCUCUC AD- A- 3836 asgsaug(Uhd)auCfUfUfuu A- 3926 VPusdAsgadGa(G2p)c UGAGAUGUAUCU 5189 1353490.1 2521276.1 gcucucsusa 2521277.1 aaadAgAfuacaucuscsg UUUGCUCUCUC AD- A- 3837 asgsaug(Uhd)AfuCfUfUfu A- 3927 VPusAfsgadGa(G2p)c UGAGAUGUAUCU 5190 1353460.1 2521217.1 ugcucucsusa 2521218.1 aaaagAfuAfcaucuscsg UUUGCUCUCUC AD- A- 3838 asgsauu(Ahd)gadGadGuu A- 3928 VPusdGsaadAudAaaa GAAGAUUAGAGA 5191 1353512.1 2521318.1 uuauuuscsa 2521319.1 cdTcdTcdTaaucususc GUUUUAUUUCU AD- A- 3839 asgsauu(Ahd)gaGfAfGfuu A- 3929 VPusdGsaadAudAaaa GAAGAUUAGAGA 5192 1353482.1 2521260.1 uuauuuscsa 2521261.1 cdTcUfcuaaucususc GUUUUAUUUCU AD- A- 3840 asgsauu(Ahd)GfaGfAfGfu A- 3930 VPusGfsaaaUfaaaacuc GAAGAUUAGAGA 5193 1353452.1 2521210.1 uuuauuuscsa 2282496.1 UfcUfaaucususc GUUUUAUUUCU AD- A- 3841 asusacagaadCgdAucga A- 3931 VPusdCsugdTa(U2p)c UGAUACAGAACG 5194 1353501.1 2521296.1 (Uhd)acasgsa 2521297.1 gaudCgdTudCuguausc AUCGAUACAGA AD- A- 3842 asusacagaaCfGfAfucga A- 3932 VPusdCsugdTa(U2p)c UGAUACAGAACG 5195 1353471.1 2521238.1 (Uhd)acasgsa 2521239.1 gaudCgUfucuguauscs AUCGAUACAGA AD- A- 3843 asusacagAfaCfGfAfucga A- 3933 VPusCfsugdTa(U2p)c UGAUACAGAACG 5196 1353441.1 2521193.1 (Uhd)acasgsa 2521194.1 gaucgUfuCfuguauscsg AUCGAUACAGA AD- A- 3844 asusgcagaudTadTgcgg A- 3934 VPusdTsugdAu(C2p)c CCAUGCAGAUUAU 5197 1353495.1 2521286.1 (Ahd)ucasasa 2521287.1 gcadTadAudCugcausg GCGGAUCAAA AD- A- 3845 asusgcagauUfAfUfgcgg A- 3935 VPusdTsugdAu(C2p)c CCAUGCAGAUUAU 5198 1353465.1 2521226.1 (Ahd)ucasasa 2521227.1 gcadTaAfucugcausgsg GCGGAUCAAA AD- A- 3846 asusgcagAfuUfAfUfgcgg A- 3936 VPusUfsugdAu(C2p)c CCAUGCAGAUUAU 5199 1353435.1 2521185.1 (Ahd)ucasasa 2521186.1 gcauaAfuCfugcausgsg GCGGAUCAAA AD- A- 3847 asusugg(Ahd)uudCgdCca A- 3937 VPusdAsaudAadAaug GAAUUGGAUUCG 5200 1353510.1 2521314.1 uuuuaususa 2521315.1 gdCgdAadTccaaususc CCAUUUUAUUU AD- A- 3848 asusugg(Ahd)uuCfGfCfca A- 3938 VPusdAsaudAadAaug GAAUUGGAUUCG 5201 1353480.1 2521256.1 uuuuaususa 2521257.1 gdCgAfauccaaususc CCAUUUUAUUU AD- A- 3849 asusugg(Ahd)UfuCfGfCfc A- 3939 VPusAfsauaAfaauggc GAAUUGGAUUCG 5202 1353450.1 2521207.1 auuuuaususa 2282456.1 gAfaUfccaaususc CCAUUUUAUUU AD- A- 3850 csasaca(Uhd)cadCcdAugc A- 3940 VPusdTsaadTc(U2p)g UCCAACAUCACCA 5203 1353492.1 2521280.1 agauusasa 2521281.1 caudGgdTgdAuguugs UGCAGAUUAU AD- A- 3851 csasaca(Uhd)caCfCfAfugc A- 3941 VPusdTsaadTc(U2p)g UCCAACAUCACCA 5204 1353462.1 2521220.1 agauusasa 2521221.1 caudGgUfgauguugsgs UGCAGAUUAU AD- A- 3852 csasaca(Uhd)CfaCfCfAfu A- 3942 VPusUfsaadTc(U2p)g UCCAACAUCACCA 5205 1353432.1 2521180.1 gcagauusasa 2521181.1 cauggUfgAfuguugsgsc UGCAGAUUAU AD- A- 3853 csasgaa(Chd)agdTcdCuua A- 3943 VPusdCsugdGa(U2p) GACAGAACAGUCC 5206 1353504.1 2521302.1 auccasgsa 2521303.1 uaagdGadCudGuucug UUAAUCCAGA AD- A- 3854 csasgaa(Chd)agUfCfCfuu A- 3944 VPusdCsugdGa(U2p) GACAGAACAGUCC 5207 1353474.1 2521244.1 aauccasgsa 2521245.1 uaagdGaCfuguucugsu UUAAUCCAGA AD- A- 3855 csasgaa(Chd)AfgUfCfCfu A- 3945 VPusCfsugdGa(U2p)u GACAGAACAGUCC 5208 1353444.1 1700826.1 uaauccasgsa 2521198.1 aaggaCfuGfuucugsusc UUAAUCCAGA AD- A- 3856 csasuca(Chd)cadTgdCaga A- 3946 VPusdGscadTadAucu AACAUCACCAUGC 5209 1353493.1 2521282.1 uuaugscsa 2521283.1 gdCadTgdGugaugsusu AGAUUAUGCG AD- A- 3857 csasuca(Chd)caUfGfCfaga A- 3947 VPusdGscadTadAucu AACAUCACCAUGC 5210 1353463.1 2521222.1 uuaugscsa 2521223.1 gdCaUfggugaugsusu AGAUUAUGCG AD- A- 3858 csasuca(Chd)CfaUfGfCfa A- 3948 VPusGfscauAfaucugc AACAUCACCAUGC 5211 1353433.1 2521182.1 gauuaugscsa 2282286.1 aUfgGfugaugsusu AGAUUAUGCG AD- A- 3859 cscsucu(Uhd)ggdAadTugg A- 3949 VPusdGscgdAadTcca UCCCUCUUGGAAU 5212 1353508.1 2521310.1 auucgscsa 2521311.1 adTudCcdAagaggsgsc UGGAUUCGCC AD- A- 3860 cscsucu(Uhd)ggAfAfUfug A- 3950 VPusdGscgdAadTcca UCCCUCUUGGAAU 5213 1353478.1 2521252.1 gauucgscsa 2521253.1 adTuCfcaagaggsgsc UGGAUUCGCC AD- A- 3861 cscsucu(Uhd)GfgAfAfUfu A- 3951 VPusGfscgaAfuccaau UCCCUCUUGGAAU 5214 1353448.1 2521204.1 ggauucgscsa 2521205.1 uCfcAfagaggsgsc UGGAUUCGCC AD- A- 3862 csusacagcadCadAcaaa A- 3952 VPusdTscadCadTuug UCCUACAGCACAA 5215 1353496.1 2521288.1 (Uhd)gugsasa 2521289.1 udTgdTgdCuguagsgsg CAAAUGUGAA AD- A- 3863 csusacagcaCfAfAfcaaa A- 3953 VPusdTscadCadTuug UCCUACAGCACAA 5216 1353466.1 2521228.1 (Uhd)gugsasa 2521229.1 udTgUfgcuguagsgsg CAAAUGUGAA AD- A- 3864 csusacagCfaCfAfAfcaaa A- 3954 VPusUfscacAfuuuguu UCCUACAGCACAA 5217 1353436.1 2521187.1 (Uhd)gugsasa 2521188.1 gUfgCfuguagsgsg CAAAUGUGAA AD- A- 3865 csusgau(Ahd)cadGadAcga A- 3955 VPusdTsaudCg(A2p)u GACUGAUACAGA 5218 1353500.1 2521294.1 ucgausasa 2521295.1 cgudTcdTgdTaucagsu ACGAUCGAUAC AD- A- 3866 csusgau(Ahd)caGfAfAfcg A- 3956 VPusdTsaudCg(A2p)u GACUGAUACAGA 5219 1353470.1 2521236.1 aucgausasa 2521237.1 cgudTcUfguaucagsusc ACGAUCGAUAC AD- A- 3867 csusgau(Ahd)CfaGfAfAfc A- 3957 VPusUfsaudCg(A2p)u GACUGAUACAGA 5220 1353440.1 1700824.1 gaucgausasa 2521192.1 cguucUfgUfaucagsusc ACGAUCGAUAC AD- A- 3868 gsasaag(Uhd)gudTudTaua A- 3958 VPusdAsccdGudAuau GAGAAAGUGUUU 5221 1334067.3 2483626.1 uacggsusa 1800443.1 adAadAcdAcuuucsusc UAUAUACGGUA AD- A- 3869 gsasaag(Uhd)guUfUfUfau A- 3959 VPusdAsccdGudAuau GAGAAAGUGUUU 5222 1353488.1 2521272.1 auacggsusa 2521273.1 adAaAfcacuuucsusc UAUAUACGGUA AD- A- 3870 gsasaag(Uhd)GfuUfUfUfa A- 3960 VPusAfsccgUfauauaa GAGAAAGUGUUU 5223 1353458.1 1700894.1 uauacggsusa 2521215.1 aAfcAfcuuucsusc UAUAUACGGUA AD- A- 3871 gsasgaa(Ahd)gudGudTuua A- 3961 VPusdCsgudAudAuaa AAGAGAAAGUGU 5224 1334065.3 2483624.1 uauacsgsa 1800396.1 adAcdAcdTuucucsusu UUUAUAUACGG AD- A- 3872 gsasgaa(Ahd)guGfUfUfuu A- 3962 VPusdCsgudAudAuaa AAGAGAAAGUGU 5225 1353487.1 2521270.1 auauacsgsa 2521271.1 adAcAfcuuucucsusu UUUAUAUACGG AD- A- 3873 gsasgaa(Ahd)GfuGfUfUfu A- 3963 VPusCfsguaUfauaaaac AAGAGAAAGUGU 5226 1353457.1 1700845.1 uauauacsgsa 2282774.1 AfcUfuucucsusu UUUAUAUACGG AD- A- 3874 gsasuucgccdAudTuuau A- 3964 VPusdGsaadAadAuaa UGGAUUCGCCAUU 5227 1353511.1 2521316.1 (Uhd)uuuscsa 2521317.1 adAudGgdCgaaucscsg UUAUUUUUCU AD- A- 3875 gsasuucgccAfUfUfuuau A- 3965 VPusdGsaadAadAuaa UGGAUUCGCCAUU 5228 1353481.1 2521258.1 (Uhd)uuuscsa 2521259.1 adAuGfgcgaaucscsg UUAUUUUUCU AD- A- 3876 gsasuucgCfcAfUfUfuuau A- 3966 VPusGfsaaaAfauaaaau UGGAUUCGCCAUU 5229 1353451.1 2521208.1 (Uhd)uuuscsa 2521209.1 GfgCfgaaucscsg UUAUUUUUCU AD- A- 3877 gsusccu(Uhd)aadTcdCaga A- 3967 VPusdCsagdGudTucu CAGUCCUUAAUCC 5230 1353507.1 2521308.1 aaccusgsa 2521309.1 gdGadTudAaggacsusg AGAAACCUGA AD- A- 3878 gsusccu(Uhd)aaUfCfCfag A- 3968 VPusdCsagdGudTucu CAGUCCUUAAUCC 5231 1353477.1 2521250.1 aaaccusgsa 2521251.1 gdGaUfuaaggacsusg AGAAACCUGA AD- A- 3879 gsusccu(Uhd)AfaUfCfCfa A- 3969 VPusCfsaggUfuucugg CAGUCCUUAAUCC 5232 1353447.1 2521203.1 gaaaccusgsa 2282416.1 aUfuAfaggacsusg AGAAACCUGA AD- A- 3880 gsusguu(Uhd)uadTadTacg A- 3970 VPusdAsagdTadCcgu AAGUGUUUUAUA 5233 1353517.1 2521328.1 guacususa 2521329.1 adTadTadAaacacsusu UACGGUACUUA AD- A- 3881 gsusguu(Uhd)uaUfAfUfac A- 3971 VPusdAsagdTadCcgu AAGUGUUUUAUA 5234 1353489.1 2521274.1 gguacususa 2521275.1 adTaUfaaaacacsusu UACGGUACUUA AD- A- 3882 gsusguu(Uhd)UfaUfAfUfa A- 3972 VPusAfsaguAfccguau AAGUGUUUUAUA 5235 1353459.1 2521216.1 cgguacususa 2282784.1 aUfaAfaacacsusu UACGGUACUUA AD- A- 3883 usasgac(Ahd)uudGcdTauu A- 3973 VPusdAsaadCadGaau AAUAGACAUUGC 5236 1353519.1 2521332.1 cuguususa 2521333.1 adGcdAadTgucuasusu UAUUCUGUUUU AD- A- 3884 usasgac(Ahd)uuGfCfUfau A- 3974 VPusdAsaadCadGaau AAUAGACAUUGC 5237 1353491.1 2521278.1 ucuguususa 2521279.1 adGcAfaugucuasusu UAUUCUGUUUU AD- A- 3885 usasgac(Ahd)UfuGfCfUfa A- 3975 VPusAfsaacAfgaauag AAUAGACAUUGC 5238 1353461.1 2521219.1 uucuguususa 2282952.1 cAfaUfgucuasusu UAUUCUGUUUU .1

TABLE 10B Exemplary Human VEGF-A siRNA Unmodified Single Strands and Duplex Sequences SEQ ID Sense SEQ ID mRNA Antisense NO: mRNA Duplex Oligo NO: Target Oligo (Anti- Target Name Name (Sense) Sense Sequence Range Name sense) Antisense Sequence Range AD- A- 3976 AAAAGAGAAAGUGU 2940- A- 4066 UTAUAAAACACTUTCT 2938- 1353514.1 2521322.1 UUUAUAA 2960 2521323.1 CUUUUCU 2960 AD- A- 3977 AAAAGAGAAAGUGU 2940- A- 4067 UTAUAAAACACTUUCU 2938- 1353484.1 2521264.1 UUUAUAA 2960 2521265.1 CUUUUCU 2960 AD- A- 3978 AAAAGAGAAAGUGU 2940- A- 4068 UUAUAAAACACUUUC 2938- 1353454.1 2521212.1 UUUAUAA 2960 2282766.1 UCUUUUCU 2960 AD- A- 3979 AAAGACUGAUACAG 1795- A- 4069 UAUCGUTCUGUAUCA 1793- 1353468.1 2521232.1 AACGAUA 1815 2521233.1 GUCUUUCC 1815 AD- A- 3980 AAAGACUGAUACAG 1795- A- 4070 UAUCGUTCUGUAUCA 1793- 1353498.1 2521292.1 AACGAUA 1815 1800369.1 GUCUUUCC 1815 AD- A- 3981 AAAGACUGAUACAG 1795- A- 4071 UAUCGUUCUGUAUCA 1793- 1353438.1 1700819.1 AACGAUA 1815 2282346.1 GUCUUUCC 1815 AD- A- 3982 AAAGAGAAAGTGUU 2941- A- 4072 UAUATAAAACACUTUC 2939- 1353515.1 2521324.1 UUAUAUA 2961 2521325.1 UCUUUUC 2961 AD- A- 3983 AAAGAGAAAGUGUU 2941- A- 4073 UAUATAAAACACUUU 2939- 1353485.1 2521266.1 UUAUAUA 2961 2521267.1 CUCUUUUC 2961 AD- A- 3984 AAAGAGAAAGUGUU 2941- A- 4074 UAUAUAAAACACUUU 2939- 1353455.1 2521213.1 UUAUAUA 2961 2282768.1 CUCUUUUC 2961 AD- A- 3985 AACAGUGCTAAUGUU 2178- A- 4075 UCCAAUAACAUTAGC 2176- 1353513.1 2521320.1 AUUGGA 2198 2521321.1 ACUGUUGG 2198 AD- A- 3986 AACAGUGCUAAUGU 2178- A- 4076 UCCAAUAACAUTAGC 2176- 1353483.1 2521262.1 UAUUGGA 2198 2521263.1 ACUGUUGG 2198 AD- A- 3987 AACAGUGCUAAUGU 2178- A- 4077 UCCAAUAACAUUAGC 2176- 1353453.1 1700832.1 UAUUGGA 2198 2521211.1 ACUGUUGG 2198 AD- A- 3988 AACGAUCGAUACAGA 1809- A- 4078 UTGGTUUCUGUAUCG 1807- 1353502.1 2521298.1 AACCAA 1829 2521299.1 AUCGUUCU 1829 AD- A- 3989 AACGAUCGAUACAGA 1809- A- 4079 UTGGTUUCUGUAUCG 1807- 1353472.1 2521240.1 AACCAA 1829 2521241.1 AUCGUUCU 1829 AD- A- 3990 AACGAUCGAUACAGA 1809- A- 4080 UUGGTUUCUGUAUCG 1807- 1353442.1 2521195.1 AACCAA 1829 2521196.1 AUCGUUCU 1829 AD- A- 3991 AAGACUGATACAGAA 1796- A- 4081 UGAUCGUUCUGTATCA 1794- 1353499.1 2483623.1 CGAUCA 1816 2521293.1 GUCUUUC 1816 AD- A- 3992 AAGACUGAUACAGA 1796- A- 4082 UGAUCGUUCUGTAUC 1794- 1353469.1 2521234.1 ACGAUCA 1816 2521235.1 AGUCUUUC 1816 AD- A- 3993 AAGACUGAUACAGA 1796- A- 4083 UGAUCGUUCUGUAUC 1794- 1353439.1 1700820.1 ACGAUCA 1816 2521191.1 AGUCUUUC 1816 AD- A- 3994 AAGAGAAAGUGUUU 2942- A- 4084 UTAUAUAAAACACTUT 2940- 1353516.1 2521326.1 UAUAUAA 2962 2521327.1 CUCUUUU 2962 AD- A- 3995 AAGAGAAAGUGUUU 2942- A- 4085 UTAUAUAAAACACUU 2940- 1353486.1 2521268.1 UAUAUAA 2962 2521269.1 UCUCUUUU 2962 AD- A- 3996 AAGAGAAAGUGUUU 2942- A- 4086 UUAUAUAAAACACUU 2940- 1353456.1 2521214.1 UAUAUAA 2962 2282770.1 UCUCUUUU 2962 AD- A- 3997 AAUUGGAUTCGCCAU 1987- A- 4087 UAUAAAAUGGCGAAU 1985- 1353509.1 2521312.1 UUUAUA 2007 2521313.1 CCAAUUCC 2007 AD- A- 3998 AAUUGGAUUCGCCAU 1987- A- 4088 UAUAAAAUGGCGAAU 1985- 1353479.1 2521254.1 UUUAUA 2007 2521255.1 CCAAUUCC 2007 AD- A- 3999 AAUUGGAUUCGCCAU 1987- A- 4089 UAUAAAAUGGCGAAU 1985- 1353449.1 2521206.1 UUUAUA 2007 2282454.1 CCAAUUCC 2007 AD- A- 4000 ACAGAACAGUCCUUA 1857- A- 4090 UTGGAUTAAGGACTGT 1855- 1353503.1 2521300.1 AUCCAA 1877 2521301.1 UCUGUCG 1877 AD- A- 4001 ACAGAACAGUCCUUA 1857- A- 4091 UTGGAUTAAGGACUG 1855- 1353473.1 2521242.1 AUCCAA 1877 2521243.1 UUCUGUCG 1877 AD- A- 4002 ACAGAACAGUCCUUA 1857- A- 4092 UUGGAUUAAGGACUG 1855- 1353443.1 2521197.1 AUCCAA 1877 2282402.1 UUCUGUCG 1877 AD- A- 4003 ACAGUCCUTAAUCCA 1862- A- 4093 UGUUTCUGGAUTAAG 1860- 1353506.1 2521306.1 GAAACA 1882 2521307.1 GACUGUUC 1882 AD- A- 4004 ACAGUCCUUAAUCCA 1862- A- 4094 UGUUTCUGGAUTAAG 1860- 1353476.1 2521248.1 GAAACA 1882 2521249.1 GACUGUUC 1882 AD- A- 4005 ACAGUCCUUAAUCCA 1862- A- 4095 UGUUTCUGGAUUAAG 1860- 1353446.1 2521201.1 GAAACA 1882 2521202.1 GACUGUUC 1882 AD- A- 4006 ACCAGGAAAGACUGA 1789- A- 4096 UCUGTATCAGUCUTUC 1787- 1353497.1 2521290.1 UACAGA 1809 2521291.1 CUGGUGC 1809 AD- A- 4007 ACCAGGAAAGACUGA 1789- A- 4097 UCUGTATCAGUCUUUC 1787- 1353467.1 2521230.1 UACAGA 1809 2521231.1 CUGGUGC 1809 AD- A- 4008 ACCAGGAAAGACUGA 1789- A- 4098 UCUGUAUCAGUCUUU 1787- 1353437.1 2521189.1 UACAGA 1809 2521190.1 CCUGGUGC 1809 AD- A- 4009 ACCAUGCAGATUAUG 1345- A- 4099 UAUCCGCAUAATCTGC 1343- 1353494.1 2521284.1 CGGAUA 1365 2521285.1 AUGGUGC 1365 AD- A- 4010 ACCAUGCAGAUUAUG 1345- A- 4100 UAUCCGCAUAATCUGC 1343- 1353464.1 2521224.1 CGGAUA 1365 2521225.1 AUGGUGC 1365 AD- A- 4011 ACCAUGCAGAUUAUG 1345- A- 4101 UAUCCGCAUAAUCUG 1343- 1353434.1 2521183.1 CGGAUA 1365 2521184.1 CAUGGUGC 1365 AD- A- 4012 AGAACAGUCCTUAAU 1859- A- 4102 UTCUGGAUUAAGGAC 1857- 1353505.1 2521304.1 CCAGAA 1879 2521305.1 TGUUCUGU 1879 AD- A- 4013 AGAACAGUCCUUAAU 1859- A- 4103 UTCUGGAUUAAGGAC 1857- 1353475.1 2521246.1 CCAGAA 1879 2521247.1 UGUUCUGU 1879 AD- A- 4014 AGAACAGUCCUUAAU 1859- A- 4104 UUCUGGAUUAAGGAC 1857- 1353445.1 2521199.1 CCAGAA 1879 2521200.1 UGUUCUGU 1879 AD- A- 4015 AGAUGUAUCUTUUGC 3073- A- 4105 UAGAGAGCAAAAGAU 3071- 1353518.1 2521330.1 UCUCUA 3093 2521331.1 ACAUCUCG 3093 AD- A- 4016 AGAUGUAUCUUUUG 3073- A- 4106 UAGAGAGCAAAAGAU 3071- 1353490.1 2521276.1 CUCUCUA 3093 2521277.1 ACAUCUCG 3093 AD- A- 4017 AGAUGUAUCUUUUG 3073- A- 4107 UAGAGAGCAAAAGAU 3071- 1353460.1 2521217.1 CUCUCUA 3093 2521218.1 ACAUCUCG 3093 AD- A- 4018 AGAUUAGAGAGUUU 2037- A- 4108 UGAAAUAAAACTCTCT 2035- 1353512.1 2521318.1 UAUUUCA 2057 2521319.1 AAUCUUC 2057 AD- A- 4019 AGAUUAGAGAGUUU 2037- A- 4109 UGAAAUAAAACTCUC 2035- 1353482.1 2521260.1 UAUUUCA 2057 2521261.1 UAAUCUUC 2057 AD- A- 4020 AGAUUAGAGAGUUU 2037- A- 4110 UGAAAUAAAACUCUC 2035- 1353452.1 2521210.1 UAUUUCA 2057 2282496.1 UAAUCUUC 2057 AD- A- 4021 AUACAGAACGAUCGA 1803- A- 4111 UCUGTAUCGAUCGTUC 1801- 1353501.1 2521296.1 UACAGA 1823 2521297.1 UGUAUCG 1823 AD- A- 4022 AUACAGAACGAUCGA 1803- A- 4112 UCUGTAUCGAUCGUU 1801- 1353471.1 2521238.1 UACAGA 1823 2521239.1 CUGUAUCG 1823 AD- A- 4023 AUACAGAACGAUCGA 1803- A- 4113 UCUGTAUCGAUCGUU 1801- 1353441.1 2521193.1 UACAGA 1823 2521194.1 CUGUAUCG 1823 AD- A- 4024 AUGCAGAUTATGCGG 1348- A- 4114 UTUGAUCCGCATAAUC 1346- 1353495.1 2521286.1 AUCAAA 1368 2521287.1 UGCAUGG 1368 AD- A- 4025 AUGCAGAUUAUGCG 1348- A- 4115 UTUGAUCCGCATAAUC 1346- 1353465.1 2521226.1 GAUCAAA 1368 2521227.1 UGCAUGG 1368 AD- A- 4026 AUGCAGAUUAUGCG 1348- A- 4116 UUUGAUCCGCAUAAU 1346- 1353435.1 2521185.1 GAUCAAA 1368 2521186.1 CUGCAUGG 1368 AD- A- 4027 AUUGGAUUCGCCAUU 1988- A- 4117 UAAUAAAAUGGCGAA 1986- 1353510.1 2521314.1 UUAUUA 2008 2521315.1 TCCAAUUC 2008 AD- A- 4028 AUUGGAUUCGCCAUU 1988- A- 4118 UAAUAAAAUGGCGAA 1986- 1353480.1 2521256.1 UUAUUA 2008 2521257.1 UCCAAUUC 2008 AD- A- 4029 AUUGGAUUCGCCAUU 1988- A- 4119 UAAUAAAAUGGCGAA 1986- 1353450.1 2521207.1 UUAUUA 2008 2282456.1 UCCAAUUC 2008 AD- A- 4030 CAACAUCACCAUGCA 1338- A- 4120 UTAATCUGCAUGGTGA 1336- 1353492.1 2521280.1 GAUUAA 1358 2521281.1 UGUUGGC 1358 AD- A- 4031 CAACAUCACCAUGCA 1338- A- 4121 UTAATCUGCAUGGUG 1336- 1353462.1 2521220.1 GAUUAA 1358 2521221.1 AUGUUGGC 1358 AD- A- 4032 CAACAUCACCAUGCA 1338- A- 4122 UUAATCUGCAUGGUG 1336- 1353432.1 2521180.1 GAUUAA 1358 2521181.1 AUGUUGGC 1358 AD- A- 4033 CAGAACAGTCCUUAA 1858- A- 4123 UCUGGAUUAAGGACU 1856- 1353504.1 2521302.1 UCCAGA 1878 2521303.1 GUUCUGUC 1878 AD- A- 4034 CAGAACAGUCCUUAA 1858- A- 4124 UCUGGAUUAAGGACU 1856- 1353474.1 2521244.1 UCCAGA 1878 2521245.1 GUUCUGUC 1878 AD- A- 4035 CAGAACAGUCCUUAA 1858- A- 4125 UCUGGAUUAAGGACU 1856- 1353444.1 1700826.1 UCCAGA 1878 2521198.1 GUUCUGUC 1878 AD- A- 4036 CAUCACCATGCAGAU 1341- A- 4126 UGCATAAUCUGCATGG 1339- 1353493.1 2521282.1 UAUGCA 1361 2521283.1 UGAUGUU 1361 AD- A- 4037 CAUCACCAUGCAGAU 1341- A- 4127 UGCATAAUCUGCAUG 1339- 1353463.1 2521222.1 UAUGCA 1361 2521223.1 GUGAUGUU 1361 AD- A- 4038 CAUCACCAUGCAGAU 1341- A- 4128 UGCAUAAUCUGCAUG 1339- 1353433.1 2521182.1 UAUGCA 1361 2282286.1 GUGAUGUU 1361 AD- A- 4039 CCUCUUGGAATUGGA 1979- A- 4129 UGCGAATCCAATUCCA 1977- 1353508.1 2521310.1 UUCGCA 1999 2521311.1 AGAGGGC 1999 AD- A- 4040 CCUCUUGGAAUUGGA 1979- A- 4130 UGCGAATCCAATUCCA 1977- 1353478.1 2521252.1 UUCGCA 1999 2521253.1 AGAGGGC 1999 AD- A- 4041 CCUCUUGGAAUUGGA 1979- A- 4131 UGCGAAUCCAAUUCC 1977- 1353448.1 2521204.1 UUCGCA 1999 2521205.1 AAGAGGGC 1999 AD- A- 4042 CUACAGCACAACAAA 1405- A- 4132 UTCACATUUGUTGTGC 1403- 1353496.1 2521288.1 UGUGAA 1425 2521289.1 UGUAGGG 1425 AD- A- 4043 CUACAGCACAACAAA 1405- A- 4133 UTCACATUUGUTGUGC 1403- 1353466.1 2521228.1 UGUGAA 1425 2521229.1 UGUAGGG 1425 AD- A- 4044 CUACAGCACAACAAA 1405- A- 4134 UUCACAUUUGUUGUG 1403- 1353436.1 2521187.1 UGUGAA 1425 2521188.1 CUGUAGGG 1425 AD- A- 4045 CUGAUACAGAACGAU 1800- A- 4135 UTAUCGAUCGUTCTGT 1798- 1353500.1 2521294.1 CGAUAA 1820 2521295.1 AUCAGUC 1820 AD- A- 4046 CUGAUACAGAACGAU 1800- A- 4136 UTAUCGAUCGUTCUGU 1798- 1353470.1 2521236.1 CGAUAA 1820 2521237.1 AUCAGUC 1820 AD- A- 4047 CUGAUACAGAACGAU 1800- A- 4137 UUAUCGAUCGUUCUG 1798- 1353440.1 1700824.1 CGAUAA 1820 2521192.1 UAUCAGUC 1820 AD- A- 4048 GAAAGUGUTUTAUAU 2946- A- 4138 UACCGUAUAUAAAAC 2944- 1334067.3 2483626.1 ACGGUA 2966 1800443.1 ACUUUCUC 2966 AD- A- 4049 GAAAGUGUUUUAUA 2946- A- 4139 UACCGUAUAUAAAAC 2944- 1353488.1 2521272.1 UACGGUA 2966 2521273.1 ACUUUCUC 2966 AD- A- 4050 GAAAGUGUUUUAUA 2946- A- 4140 UACCGUAUAUAAAAC 2944- 1353458.1 1700894.1 UACGGUA 2966 2521215.1 ACUUUCUC 2966 AD- A- 4051 GAGAAAGUGUTUUA 2944- A- 4141 UCGUAUAUAAAACAC 2942- 1334065.3 2483624.1 UAUACGA 2964 1800396.1 TUUCUCUU 2964 AD- A- 4052 GAGAAAGUGUUUUA 2944- A- 4142 UCGUAUAUAAAACAC 2942- 1353487.1 2521270.1 UAUACGA 2964 2521271.1 UUUCUCUU 2964 AD- A- 4053 GAGAAAGUGUUUUA 2944- A- 4143 UCGUAUAUAAAACAC 2942- 1353457.1 1700845.1 UAUACGA 2964 2282774.1 UUUCUCUU 2964 AD- A- 4054 GAUUCGCCAUTUUAU 1992- A- 4144 UGAAAAAUAAAAUGG 1990- 1353511.1 2521316.1 UUUUCA 2012 2521317.1 CGAAUCCG 2012 AD- A- 4055 GAUUCGCCAUUUUAU 1992- A- 4145 UGAAAAAUAAAAUGG 1990- 1353481.1 2521258.1 UUUUCA 2012 2521259.1 CGAAUCCG 2012 AD- A- 4056 GAUUCGCCAUUUUAU 1992- A- 4146 UGAAAAAUAAAAUGG 1990- 1353451.1 2521208.1 UUUUCA 2012 2521209.1 CGAAUCCG 2012 AD- A- 4057 GUCCUUAATCCAGAA 1865- A- 4147 UCAGGUTUCUGGATU 1863- 1353507.1 2521308.1 ACCUGA 1885 2521309.1 AAGGACUG 1885 AD- A- 4058 GUCCUUAAUCCAGAA 1865- A- 4148 UCAGGUTUCUGGAUU 1863- 1353477.1 2521250.1 ACCUGA 1885 2521251.1 AAGGACUG 1885 AD- A- 4059 GUCCUUAAUCCAGAA 1865- A- 4149 UCAGGUUUCUGGAUU 1863- 1353447.1 2521203.1 ACCUGA 1885 2282416.1 AAGGACUG 1885 AD- A- 4060 GUGUUUUATATACGG 2950- A- 4150 UAAGTACCGUATATAA 2948- 1353517.1 2521328.1 UACUUA 2970 2521329.1 AACACUU 2970 AD- A- 4061 GUGUUUUAUAUACG 2950- A- 4151 UAAGTACCGUATAUA 2948- 1353489.1 2521274.1 GUACUUA 2970 2521275.1 AAACACUU 2970 AD- A- 4062 GUGUUUUAUAUACG 2950- A- 4152 UAAGUACCGUAUAUA 2948- 1353459.1 2521216.1 GUACUUA 2970 2282784.1 AAACACUU 2970 AD- A- 4063 UAGACAUUGCTAUUC 3365- A- 4153 UAAACAGAAUAGCAA 3363- 1353519.1 2521332.1 UGUUUA 3385 2521333.1 TGUCUAUU 3385 AD- A- 4064 UAGACAUUGCUAUUC 3365- A- 4154 UAAACAGAAUAGCAA 3363- 1353491.1 2521278.1 UGUUUA 3385 2521279.1 UGUCUAUU 3385 AD- A- 4065 UAGACAUUGCUAUUC 3365- A- 4155 UAAACAGAAUAGCAA 3363- 1353461.1 2521219.1 UGUUUA 3385 2282952.1 UGUCUAUU 3385

TABLE 18A Exemplary Human VEGF-A siRNA Modified Single Strands and Duplex Sequences SEQ Anti- ID SEQ ID Sense SEQ ID sense NO: NO: Duplex Oligo NO: Oligo (Anti- mRNA Target (mRNA Name Name (Sense) Sense Sequence Name sense) Antisense Sequence Sequence target) AD- A- 4164 csgsaca(Ghd)AfaCfAfGf A- 4176 VPusGfsauua(Agn)ggac AUCGACAGAACA 4188 1020574 1110770.1 uccuuaauscsa 1701268.1 ugUfuCfugucgsasu GUCCUUAAUCC AD- A- 4165 csasgaa(Chd)AfgUfCfCf A- 4177 VPusCfsuggAfuUfAfag GACAGAACAGUC 4189 901094 1700826.1 uuaauccasgsa 1068918.1 gaCfuGfuucugsusc CUUAAUCCAGA AD- A- 4166 csasgaa(Chd)AfgUfCfCf A- 4178 VPusCfsugga(Tgn)uaag GACAGAACAGUC 4190 1020575 1700826.1 uuaauccasgsa 1701270.1 gaCfuGfuucugsusc CUUAAUCCAGA AD- A- 4167 asascag(Uhd)GfcUfAfAf A- 4179 VPusCfscaaUfaAfCfauu UUAACAGUGCUA 4191 901100 1700832.1 uguuauugsgsa 1069342.1 aGfcAfcuguusasa AUGUUAUUGGU AD- A- 4168 asgsugc(Uhd)AfaUfGfUf A- 4180 VPusAfscacCfaAfUfaac ACAGUGCUAAUG 4192 901101 1700833.1 uauuggugsusa 1069348.1 aUfuAfgcacusgsu UUAUUGGUGUC AD- A- 4169 gsasgaa(Ahd)GfuGfUfUf A- 4181 VPusCfsguaUfaUfAfaaa AAGAGAAAGUGU 4193 901113 1700845.1 uuauauacsgsa 1070290.1 cAfcUfuucucsusu UUUAUAUACGG AD- A- 4170 asasaau(Ahd)GfaCfAfUf A- 4182 VPusAfsgaaUfaGfCfaau AUAAAAUAGACA 4194 901123 1700855.1 ugcuauucsusa 1070790.1 gUfcUfauuuusasu UUGCUAUUCUG AD- A- 4171 asasaua(Ghd)AfcAfUfUf A- 4183 VPusCfsagaAfuAfGfcaa UAAAAUAGACAU 4195 901124 1700856.1 gcuauucusgsa 1070792.1 uGfuCfuauuususa UGCUAUUCUGU AD- A- 4172 gsasaag(Uhd)GfuUfUfUf A- 4184 VPusAfsccgUfaUfAfuaa GAGAAAGUGUUU 4196 901158 1700894.1 auauacggsusa 1070294.1 aAfcAfcuuucsusc UAUAUACGGUA AD- A- 4173 gsusuuu(Ahd)UfaUfAfCf A- 4185 VPusAfsuaaGfuAfCfcgu GUGUUUUAUAUA 4197 901159 1700895.1 gguacuuasusa 1070306.1 aUfaUfaaaacsasc CGGUACUUAUU AD- A- 4174 asgsugc(Uhd)aadTgdTua A- 4186 VPusdAscadCcdAauaad ACAGUGCUAAUG 4198 1020573 1890520.1 uuggugsusa 1800384.1 CadTudAgcacusgsu UUAUUGGUGUC AD- A- 4175 asasaau(Ahd)gadCadTug A- 4187 VPusdAsgadAudAgcaad AUAAAAUAGACA 4199 1023143 1895607.1 cuauucsusa 1800407.1 TgdTcdT auuuus asu UUGCUAUUCUG

TABLE 18B Exemplary Human VEGF-A siRNA Unmodified Single Strands and Duplex Sequences SEQ ID mRNA Sense SEQ ID mRNA Antisense NO: Duplex Oligo NO: Target Oligo (Anti- Antisense Target Name Name (Sense) Sense Sequence Range Name sense) Sequence Range AD- A- 4200 CGACAGAACAGUCCU 1855- A- 4212 UGAUUAAGGACUGUU 1853-1875 1020574 1110770.1 UAAUCA 1875 1701268.1 CUGUCGAU AD- A- 4201 CAGAACAGUCCUUAA 1858- A- 4213 UCUGGAUUAAGGACU 1856-1878 901094 1700826.1 UCCAGA 1878 1068918.1 GUUCUGUC AD- A- 4202 CAGAACAGUCCUUAA 1858- A- 4214 UCUGGATUAAGGACU 1856-1878 1020575 1700826.1 UCCAGA 1878 1701270.1 GUUCUGUC AD- A- 4203 AACAGUGCUAAUGU 2178- A- 4215 UCCAAUAACAUUAGC 2176-2198 901100 1700832.1 UAUUGGA 2198 1069342.1 ACUGUUAA AD- A- 4204 AGUGCUAAUGUUAU 2181- A- 4216 UACACCAAUAACAUU 2179-2201 901101 1700833.1 UGGUGUA 2201 1069348.1 AGCACUGU AD- A- 4205 GAGAAAGUGUUUUA 2944- A- 4217 UCGUAUAUAAAACAC 2942-2964 901113 1700845.1 UAUACGA 2964 1070290.1 UUUCUCUU AD- A- 4206 AAAAUAGACAUUGC 3361- A- 4218 UAGAAUAGCAAUGUC 3359-3381 901123 1700855.1 UAUUCUA 3381 1070790.1 UAUUUUAU AD- A- 4207 AAAUAGACAUUGCU 3362- A- 4219 UCAGAAUAGCAAUGU 3360-3382 901124 1700856.1 AUUCUGA 3382 1070792.1 CUAUUUUA AD- A- 4208 GAAAGUGUUUUAUA 2946- A- 4220 UACCGUAUAUAAAAC 2944-2966 901158 1700894.1 UACGGUA 2966 1070294.1 ACUUUCUC AD- A- 4209 GUUUUAUAUACGGU 2952- A- 4221 UAUAAGUACCGUAUA 2950-2972 901159 1700895.1 ACUUAUA 2972 1070306.1 UAAAACAC AD- A- 4210 AGUGCUAATGTUAUU 2181- A- 4222 UACACCAAUAACATU 2179-2201 1020573 1890520.1 GGUGUA 2201 1800384.1 AGCACUGU AD- A- 4211 AAAAUAGACATUGCU 3361- A- 4223 UAGAAUAGCAATGTCT 3359-3381 1023143 1895607.1 AUUCUA 3381 1800407.1 AUUUUAU

Example 2. In Vitro Screening of VEGF-A siRNA Experimental Methods

Cell Culture and Transfections:

Cos 7 Cell Transfections

Cos-7 (ATCC) were transfected by adding 5 μl of 1 ng/μl psiCHECK2 vector (Blue Heron Biotechnology) containing either Cynomolgus monkey (XM_005552887) or mouse (NM_001025250), 4.9 μl of Opti-MEM, 0.1 μl of Lipofectamine 2000 (Invitrogen, Carlsbad Calif. cat #11668-019), and 5 μl of siRNA duplexes per well into a 384-well plate. Following a 15-minute incubation at room temperature, thirty-five μl of Dulbecco's Modified Eagle Medium (ThermoFisher) containing ˜5×10³ cells were then added to the siRNA-transfection mixture. Cells were incubated for 48 hours followed by Firefly (transfection control) and Renilla (fused to target sequence) luciferase measurements. Experiments were performed at 10 nM, 1 nM, and 0.1 nM.

APRE-19 Cell, hTERT REP-1, and Primary Human Hepatocyte Cell Transfections

ARPE-19 cells, hTERT RPE-1, or primary human hepatocyte cells (ATCC) were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of RNAiMAX per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well, with 4 replicates of each siRNA duplex, into a 384-well plate, and incubated at room temperature for 15 minutes. Forty μl of DMEM:F12 Medium (ThermoFisher) containing ˜5×10³ cells were then added to the siRNA-transfection mixture. Cells were incubated for 24 hours prior to RNA purification. Experiments were performed at 50 nM, 10 nM, 1 nM, and 0.1 nM.

Free Uptake Transfection:

Cryopreserved primary human hepatocytes were thawed at 37° C. in a water bath immediately prior to usage and re-suspended at 0.26×10⁶ cells/mL in InVitroGRO CP (plating) medium (Celsis In Vitro Technologies, catalog number Z99029). During transfections, cells were plated onto a BD BioCoat 96 well collagen plate (BD, 356407) at 25,000 cells per well and incubated at 37° C. in an atmosphere of 5% CO2. Free Uptake experiments were performed by adding 10 μL of siRNA duplexes in PBS per well into a 96 well plate. Ninety μL of complete growth media containing appropriate cell number for the cell was then added to the siRNA. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 500 nM, 100 nM, 10 nM, and 1 nM final duplex.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit:

RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 70 μl of Lysis/Binding Buffer and 10 μl of lysis buffer containing 3 μl of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 150 μl Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 μl Elution Buffer, re-captured and supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813):

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25×dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H2O per reaction was added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 h 37° C.

Real Time PCR:

Two μl of cDNA and 50 Lightcycler 480 probe master mix (Roche Cat #04887301001) were added to either 0.5 μl of Human GAPDH TaqMan Probe (4326317E) and 0.5 μl VEGFA Human probe (Hs00900055_m1, Thermo) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche). Each duplex was tested at least two times and data were normalized to cells transfected with a non-targeting control siRNA. To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with a non-targeting control siRNA.

Results

The results of the multi-dose screen in human retinal pigment epithelial cells (ARPE-19) and human hTERT-immortalized retinal pigment epithelial cells (hTERT RPE-1) with three sets of exemplary human VEGF-A siRNAs are shown in Table 6A (correspond to siRNAs in Table 2A and Table 2B), Table 6B (correspond to siRNAs in Table 3A and Table 3B), and 6C (correspond to siRNAs in Table 4A and Table 4B). The multi-dose experiments were performed at 50 nM, 10 nM, 1 nM, and 0.1 nM final duplex concentrations and the data are expressed as percent message remaining relative to non-targeting control. Of the exemplary siRNA duplexes evaluated, 28 achieved a knockdown of VEGF-A of >90%, 108 achieved a knockdown of

VEGF-A of ≥60%, and 229 achieved a knockdown of VEGF-A of ≥30% in in ARPE-19 cells when administered at the 10 nM concentration.

TABLE 6A VEGF-A endogenous in vitro multi-dose screen with one set of exemplary human VEGF-A siRNAs ARPE-19 hTERT RPE-1 50 10 1 0.1 50 10 1 0.1 Sample_Name nM StDev nM StDev nM StDev nM StDev nM StDev nM StDev nM StDev nM StDev AD-901349.1 29.9 4.1 24.7 3.0 34.0 3.0 54.3 5.7 26.5 7.9 26.0 1.7 64.1 11.3 94.0 17.7 AD-901376.1 28.3 4.6 25.4 7.6 35.7 8.8 50.3 14.1 15.1 2.7 20.9 4.1 37.6 8.3 46.9 4.6 AD-901356.1 30.6 2.9 27.9 2.8 36.5 2.5 60.3 7.6 21.0 3.5 25.3 9.7 39.3 8.9 91.6 12.5 AD-901355.1 42.0 2.2 28.7 3.7 39.6 0.2 65.7 11.5 26.9 2.3 27.4 9.6 44.8 4.0 92.6 22.9 AD-901407.1 27.8 11.7 30.9 4.0 55.4 6.2 106.1 19.2 39.0 7.9 41.1 12.4 69.5 10.9 87.5 21.1 AD-901367.1 39.0 4.8 31.5 3.7 38.6 7.1 50.4 2.2 31.0 6.3 39.3 5.5 38.1 5.2 77.0 13.4 AD-901352.1 42.7 1.8 34.6 6.1 48.5 6.6 57.4 4.9 27.1 7.7 28.9 4.5 51.6 10.4 76.8 15.3 AD-901348.1 44.5 7.3 35.0 7.2 40.6 4.7 62.4 9.1 35.4 6.8 23.0 4.2 50.0 18.5 97.9 21.6 AD-901354.1 45.0 3.6 35.9 7.1 40.9 2.1 61.2 4.0 34.1 8.9 27.0 6.2 34.7 8.7 81.9 18.7 AD-901353.1 50.8 5.9 37.6 8.6 29.8 4.7 52.4 2.4 36.1 6.5 21.5 4.3 40.4 8.7 98.2 11.7 AD-901375.1 44.5 7.1 40.2 2.0 43.0 6.6 60.0 12.2 30.5 1.8 30.4 3.3 46.9 24.4 61.3 4.9 AD-901345.1 53.1 6.0 42.9 9.0 74.3 13.7 73.0 16.3 74.8 12.7 62.2 18.1 61.7 9.5 85.2 14.5 AD-901357.1 39.8 11.7 42.9 1.1 54.2 3.3 78.1 14.9 37.2 7.4 42.3 15.4 43.2 2.9 97.9 19.8 AD-901334.1 55.7 4.7 43.4 7.2 77.4 9.8 76.7 6.0 74.9 18.4 47.9 12.8 73.3 17.2 85.9 8.5 AD-901313.1 32.9 3.6 44.2 9.8 77.8 17.5 64.1 7.0 39.7 12.8 47.9 5.8 101.3 4.5 69.6 18.2 AD-901344.1 71.9 5.4 46.9 6.7 78.6 16.2 59.4 9.0 75.8 6.0 72.9 21.0 62.0 13.2 78.4 7.8 AD-901366.1 56.1 9.1 47.2 7.4 61.9 5.7 69.1 6.6 42.5 11.3 49.6 12.8 58.3 14.2 97.4 18.8 AD-901337.1 70.7 7.9 47.7 9.4 101.6 21.1 82.6 9.4 91.8 17.2 64.0 25.7 80.8 21.3 86.4 7.4 AD-901335.1 58.7 9.2 48.0 7.7 109.6 32.9 84.5 9.8 75.6 4.6 62.1 15.5 72.2 19.6 92.4 18.7 AD-901398.1 51.6 2.2 48.0 8.3 63.3 5.6 91.1 11.3 39.6 8.2 55.7 9.8 71.8 10.1 56.2 8.4 AD-901314.1 43.1 10.7 48.7 10.8 94.3 25.4 99.6 15.1 49.9 6.0 50.9 10.9 67.9 15.4 100.9 31.8 AD-901386.1 51.7 6.0 49.5 2.7 82.7 59.5 75.9 21.6 62.6 20.0 42.9 8.8 55.4 20.7 45.8 8.9 AD-901336.1 67.6 5.1 51.4 12.8 88.0 14.9 74.3 9.5 88.1 10.2 51.7 16.0 77.8 19.7 85.0 16.0 AD-901310.1 40.9 8.2 53.0 5.6 88.6 18.3 82.0 5.8 46.8 7.6 52.4 10.7 71.9 11.7 76.1 7.3 AD-901321.1 41.4 7.4 53.1 13.3 87.7 15.2 67.0 16.0 36.4 8.2 40.2 4.3 87.2 13.0 89.2 8.0 AD-901382.1 57.5 4.2 53.7 3.0 62.8 4.5 90.1 19.4 41.0 12.4 37.9 9.3 68.2 10.5 63.9 9.5 AD-901384.1 49.7 6.8 53.8 12.8 54.0 12.3 87.8 38.9 43.5 5.7 52.6 5.1 60.1 9.4 56.1 12.1 AD-901339.1 80.3 16.6 54.0 7.4 76.7 11.8 74.7 3.5 96.3 18.7 68.3 12.5 68.9 13.3 89.7 11.8 AD-901363.1 68.5 11.9 55.2 9.3 54.3 4.3 71.2 8.6 52.2 5.7 54.8 12.0 45.5 1.5 86.0 15.8 AD-901325.1 71.6 2.4 55.6 7.1 111.4 11.4 95.5 4.4 77.5 15.7 69.8 10.8 88.4 10.0 96.8 6.0 AD-901350.1 60.4 9.5 56.5 8.4 66.9 6.6 73.3 3.1 50.7 6.7 45.6 3.9 85.0 8.9 92.4 12.9 AD-901365.1 68.6 5.1 56.5 6.6 64.8 7.6 79.6 12.3 48.9 18.5 52.3 13.3 56.8 16.7 96.1 8.8 AD-901306.1 55.1 12.5 57.7 5.8 96.6 17.2 97.3 24.5 66.5 10.5 74.7 16.8 75.6 9.7 108.6 25.5 AD-901361.1 50.8 6.9 58.5 18.8 45.6 3.1 68.6 10.3 44.9 4.3 36.2 9.9 57.7 6.5 95.4 14.9 AD-901320.1 52.3 11.4 60.0 5.7 94.3 23.4 69.8 3.8 48.1 19.3 48.3 3.8 68.9 13.2 84.4 18.8 AD-901405.1 64.5 3.4 60.5 9.8 80.4 7.5 83.6 15.4 63.1 9.2 72.3 21.9 74.6 9.3 55.6 6.4 AD-901338.1 52.7 5.1 61.7 4.1 82.0 12.2 86.9 13.2 73.4 8.4 60.8 20.1 58.8 9.8 103.1 16.6 AD-901383.1 62.0 8.9 62.2 7.7 78.2 11.8 92.7 14.8 57.0 9.8 62.6 11.9 75.1 16.6 75.2 7.0 AD-901333.1 82.0 7.2 62.5 12.2 94.1 17.3 85.2 6.9 92.2 5.4 66.8 12.1 79.2 15.9 70.5 37.8 AD-901330.1 75.7 8.7 63.0 9.7 96.4 6.3 104.5 23.5 90.8 8.4 63.2 16.4 82.5 16.6 109.3 30.1 AD-901360.1 61.4 8.8 64.0 10.3 59.9 6.5 90.6 32.4 77.5 3.7 63.7 6.4 64.1 14.7 95.1 14.9 AD-901358.1 67.4 11.4 65.3 5.4 71.0 3.6 82.3 6.7 58.5 5.3 49.5 6.5 62.8 4.6 94.4 11.9 AD-901406.1 67.6 7.4 65.6 7.7 77.2 6.7 93.5 2.0 63.3 13.4 65.2 5.7 69.4 9.5 86.9 30.3 AD-901326.1 78.8 14.8 65.8 16.8 120.8 26.0 88.1 8.0 85.9 5.7 69.6 11.8 88.6 15.2 98.3 14.6 AD-901377.1 47.2 7.3 65.8 11.4 57.5 12.7 68.0 22.8 42.8 6.2 47.4 6.3 54.8 10.2 63.4 15.1 AD-901351.1 69.4 9.6 66.9 10.1 82.5 6.8 85.7 5.3 73.9 8.0 56.8 13.1 81.2 30.7 97.6 36.5 AD-901415.1 77.8 13.7 68.1 18.4 78.6 14.7 119.6 45.3 85.6 6.4 57.2 10.5 83.8 7.7 78.5 19.4 AD-901342.1 87.4 20.2 70.1 10.3 113.4 21.8 79.3 12.1 90.5 27.6 56.2 11.2 80.2 21.4 91.5 14.4 AD-901420.1 61.1 7.0 71.0 5.7 75.3 4.0 107.9 37.3 52.4 2.8 43.5 7.9 67.8 13.7 80.8 8.9 AD-901312.1 62.1 10.7 71.2 8.0 102.4 22.9 76.5 2.4 75.1 7.7 60.1 15.4 100.2 30.7 73.8 17.5 AD-901340.1 88.8 12.8 71.6 10.3 100.4 19.9 93.3 12.4 94.1 31.9 69.3 11.0 87.6 24.7 100.6 10.9 AD-901392.1 49.7 9.0 71.6 13.2 69.0 4.0 81.0 47.1 41.3 2.9 61.4 10.0 77.7 12.1 74.3 5.4 AD-901327.1 72.4 15.6 71.8 8.7 114.4 13.3 80.6 2.7 89.8 13.6 75.7 12.3 82.7 23.2 92.0 8.8 AD-901328.1 77.7 13.0 72.6 6.3 104.1 10.6 76.9 7.7 87.2 16.3 70.5 4.1 89.5 8.4 86.4 10.7 AD-901370.1 65.9 6.5 72.9 7.8 79.9 6.7 70.6 4.4 67.8 9.5 81.6 5.6 70.3 16.6 52.8 35.3 AD-901399.1 63.2 10.4 72.9 7.8 79.3 5.9 98.0 11.8 52.6 4.9 78.5 18.2 62.3 12.7 68.9 7.0 AD-901359.1 83.7 13.3 73.4 12.1 66.6 8.8 69.4 7.1 80.2 20.5 63.3 20.6 70.5 6.9 90.7 19.1 AD-901373.1 81.5 10.9 73.4 4.6 73.5 2.4 69.8 8.7 52.1 11.0 61.9 17.9 60.7 10.9 56.7 12.5 AD-901332.1 79.6 17.9 73.7 4.9 123.3 17.1 93.6 6.1 95.2 13.6 56.4 15.2 96.5 7.9 89.0 14.4 AD-901311.1 53.7 10.4 74.5 9.5 98.0 27.9 72.0 4.0 67.5 16.0 69.0 9.2 64.6 10.3 73.9 3.3 AD-901423.1 61.8 10.6 74.5 9.5 72.3 8.7 104.0 24.4 48.8 3.6 56.4 10.5 68.3 12.8 73.5 8.2 AD-901374.1 92.4 18.4 74.5 9.6 86.9 12.5 71.0 14.7 90.5 13.9 52.7 13.4 81.5 12.0 61.3 12.7 AD-901319.1 73.3 19.5 75.1 6.3 126.0 19.4 77.4 7.6 88.5 16.0 81.3 7.2 64.0 20.9 93.5 33.9 AD-901341.1 88.8 12.2 75.5 8.1 107.3 10.6 79.2 5.8 77.2 20.8 86.1 24.0 96.8 15.9 93.0 10.6 AD-901422.1 51.3 3.2 75.8 5.2 55.6 5.8 95.6 25.5 40.2 4.1 39.9 9.4 61.8 15.0 76.2 19.9 AD-901385.1 67.7 13.9 75.8 16.1 86.8 10.9 93.1 21.7 72.9 11.6 72.2 14.5 76.4 13.2 65.1 10.3 AD-901391.1 72.0 11.9 76.2 8.8 82.3 12.0 76.8 22.5 71.6 14.7 63.4 16.7 81.2 6.6 85.9 13.3 AD-901329.1 72.5 10.6 76.3 11.1 107.1 12.2 79.8 9.2 98.2 17.4 75.5 10.9 100.5 15.9 93.9 6.9 AD-901331.1 101.1 11.5 77.9 13.7 120.0 24.9 84.0 10.0 126.3 25.1 76.3 19.3 82.2 6.1 79.0 22.0 AD-901368.1 73.7 9.1 79.5 11.1 82.4 5.5 90.1 31.1 98.3 21.8 97.4 7.8 64.4 19.9 85.4 4.9 AD-901364.1 82.5 10.9 79.5 16.7 75.7 8.9 81.4 2.6 65.8 7.1 65.3 18.7 48.4 7.4 90.9 5.5 AD-901389.1 69.1 10.2 80.3 6.1 80.4 9.2 87.2 22.8 50.7 2.4 55.9 6.4 82.8 21.5 59.0 16.1 AD-901421.1 54.9 7.1 80.9 6.9 63.4 9.2 96.5 25.7 50.4 9.2 37.8 3.8 70.9 15.3 61.1 11.7 AD-901380.1 85.6 16.8 81.0 6.5 60.9 9.0 83.7 9.2 74.9 12.3 77.7 6.6 54.5 11.3 75.8 19.3 AD-901343.1 102.8 17.6 81.8 13.7 127.7 32.4 99.8 11.8 133.1 18.9 81.7 20.5 93.1 6.4 81.8 13.4 AD-901317.1 89.8 12.2 81.8 4.8 131.2 51.1 90.6 5.2 120.7 22.3 86.4 4.5 69.7 11.0 76.7 7.1 AD-901424.1 62.9 4.8 82.2 14.8 64.9 10.2 86.0 8.1 68.7 13.3 63.1 22.6 76.5 15.9 71.8 24.4 AD-901431.1 84.8 11.4 82.9 20.5 75.7 10.5 96.4 29.5 79.4 15.9 88.8 25.2 85.1 8.9 80.8 21.7 AD-901378.1 71.4 5.4 82.9 5.5 77.3 8.5 80.6 9.3 56.8 14.3 40.8 13.4 82.0 24.4 78.9 14.1 AD-901434.1 70.3 11.3 83.8 8.4 59.4 1.3 92.1 11.3 62.6 8.3 72.7 17.5 69.9 12.0 82.0 11.3 AD-901412.1 83.4 15.9 84.5 32.2 94.4 4.4 81.6 19.4 82.3 3.9 50.8 5.2 85.6 21.9 74.5 8.0 AD-901426.1 96.7 7.7 85.1 13.5 68.2 7.2 104.4 9.3 92.5 15.3 61.5 4.0 56.0 7.0 80.4 15.0 AD-901322.1 74.9 14.5 85.5 12.4 118.1 15.9 117.4 22.0 107.2 20.0 92.5 15.9 98.9 16.0 107.3 8.5 AD-901381.1 92.6 10.5 85.7 4.9 93.9 10.2 90.8 17.0 72.5 15.0 74.0 16.8 85.0 21.6 71.5 16.1 AD-901324.1 93.0 10.1 87.3 14.3 142.1 25.2 92.9 5.5 133.9 8.1 94.9 11.1 94.3 9.2 91.4 4.7 AD-901347.1 84.2 11.6 87.7 9.5 101.8 19.4 83.9 4.4 96.1 10.3 84.6 25.5 105.8 6.4 96.3 13.4 AD-901379.1 81.1 5.9 87.8 6.2 72.1 6.9 80.4 6.7 79.3 7.6 59.9 15.5 82.5 18.4 59.1 18.7 AD-901428.1 71.8 4.4 88.2 18.3 74.5 8.0 93.0 16.6 89.7 13.0 52.5 12.6 65.9 17.7 76.7 13.3 AD-901371.1 75.5 7.5 88.2 15.6 77.3 5.7 82.5 5.9 71.7 8.5 56.8 12.0 68.6 10.7 71.2 20.0 AD-901408.1 74.4 14.1 88.8 30.8 91.5 5.1 85.5 11.2 75.4 14.5 57.2 15.1 97.4 21.5 76.5 13.3 AD-901417.1 94.8 14.8 89.2 14.0 101.7 5.9 117.2 33.3 85.4 23.1 76.9 12.4 92.6 6.8 76.2 21.6 AD-901400.1 82.9 10.1 89.7 8.6 97.9 11.3 97.7 22.9 97.5 12.8 101.9 12.3 98.8 13.0 65.3 17.5 AD-901323.1 74.6 14.8 90.4 12.0 130.1 25.7 86.8 3.3 101.8 5.7 93.7 18.2 80.4 12.4 96.3 20.5 AD-901316.1 91.0 18.0 90.8 8.3 139.5 42.5 96.3 3.1 99.6 17.7 93.6 8.8 80.1 28.4 96.5 18.2 AD-901315.1 86.7 14.1 90.8 6.4 130.0 40.3 92.6 4.0 125.3 13.0 107.4 11.9 72.7 22.2 97.5 7.6 AD-901395.1 77.9 11.7 90.9 5.9 80.0 4.0 86.9 20.7 65.9 13.2 63.8 10.5 87.3 8.0 74.9 19.2 AD-901318.1 78.6 6.2 92.1 11.8 131.0 50.7 90.2 11.5 112.5 21.6 91.5 12.2 73.0 12.4 83.6 15.4 AD-901390.1 75.3 12.2 93.3 5.9 95.8 9.4 96.1 23.2 47.0 6.9 66.4 7.4 71.5 4.7 64.3 14.3 AD-901387.1 91.5 9.4 94.9 6.0 100.0 18.9 93.5 10.9 131.7 17.8 89.3 10.7 89.1 17.7 69.4 13.4 AD-901307.1 66.0 13.3 95.4 11.1 105.1 21.0 90.1 4.9 119.0 15.4 100.5 12.7 74.3 28.2 105.8 10.9 AD-901410.1 91.1 15.6 96.5 28.2 104.3 15.4 91.4 7.4 93.9 9.4 55.3 10.6 98.9 32.4 79.6 13.4 AD-901433.1 86.9 4.6 96.9 18.9 74.6 2.3 98.2 8.3 81.1 19.2 58.0 15.9 81.0 12.6 73.6 12.0 AD-901308.1 67.0 5.1 97.7 18.4 136.0 31.1 89.2 5.3 116.8 18.1 109.3 4.6 99.4 12.4 80.8 8.5 AD-901414.1 88.8 12.1 98.1 31.3 94.5 2.6 94.0 16.9 88.5 4.8 64.8 17.5 70.2 8.3 82.3 11.3 AD-901309.1 67.5 6.9 98.7 12.4 124.9 29.1 91.8 3.9 111.9 21.4 84.7 12.8 97.9 16.1 88.3 18.1 AD-901362.1 75.1 11.6 99.1 22.7 91.0 7.1 91.6 12.6 93.3 9.1 99.8 38.1 69.3 19.5 92.9 13.0 AD-901397.1 86.1 9.0 99.3 8.7 94.9 5.6 88.4 19.5 99.2 14.2 119.6 15.6 83.0 11.1 67.2 17.1 AD-901419.1 74.3 12.2 100.1 11.9 80.7 8.0 116.8 34.6 74.5 14.5 49.9 4.4 89.7 14.6 72.7 12.1 AD-901413.1 92.5 12.3 101.4 37.9 97.6 3.4 96.1 18.3 77.1 5.9 59.3 11.9 75.0 18.1 67.4 2.0 AD-901401.1 95.3 6.8 101.8 10.4 116.3 16.9 98.8 24.7 138.0 19.8 97.0 15.7 102.4 24.1 81.1 10.2 AD-901411.1 83.9 8.7 102.6 36.0 93.3 8.6 105.0 4.3 79.2 14.1 55.2 8.4 80.4 7.4 75.1 28.5 AD-901372.1 85.7 9.4 103.1 18.7 89.3 4.3 89.9 12.5 100.5 7.7 97.0 20.4 73.8 9.6 47.5 4.4 AD-901425.1 86.6 13.9 104.1 15.8 100.2 12.0 110.9 21.8 88.0 11.6 66.1 22.7 87.1 20.1 74.9 17.6 AD-901409.1 110.5 16.6 106.3 26.4 112.3 10.1 98.9 9.1 102.0 12.9 53.7 18.0 90.5 8.8 87.0 24.0 AD-901418.1 91.4 17.7 106.6 12.5 104.2 5.8 115.4 41.5 84.9 16.8 58.5 14.0 94.1 2.4 79.4 3.5 AD-901393.1 95.9 14.4 107.2 19.2 104.5 7.5 79.9 14.8 95.8 15.0 101.9 28.1 94.8 18.2 65.6 22.0 AD-901388.1 83.2 14.1 108.9 4.0 102.2 9.0 96.5 10.3 69.7 4.2 93.0 12.0 90.5 5.4 63.6 22.4 AD-901404.1 99.7 7.5 110.2 6.7 119.3 24.1 94.8 6.8 130.3 22.2 108.2 6.2 83.6 13.6 84.4 9.4 AD-901346.1 78.3 11.7 110.7 28.7 97.5 15.9 70.1 4.8 89.4 8.0 75.4 20.2 76.7 24.4 105.1 10.9 AD-901403.1 92.7 11.1 111.4 9.2 101.5 5.3 96.1 9.3 94.2 13.5 111.6 19.8 79.3 7.0 62.3 6.4 AD-901396.1 95.6 12.6 112.1 20.6 101.1 10.1 97.7 11.4 88.7 6.9 116.5 36.0 89.3 3.2 79.8 22.4 AD-901432.1 86.4 16.0 113.4 16.6 68.0 6.3 97.4 15.6 98.3 13.9 67.9 6.9 81.5 17.3 77.7 17.1 AD-901435.1 93.4 6.6 115.4 12.9 82.5 12.0 97.1 10.8 100.4 9.0 68.1 31.6 82.2 25.7 78.0 11.7 AD-901416.1 103.4 18.0 116.6 18.3 110.7 16.3 98.3 27.8 102.4 25.4 77.5 16.6 100.2 4.4 82.1 22.3 AD-901394.1 106.8 12.4 118.1 11.0 111.8 9.5 92.1 16.4 137.8 38.9 117.2 37.8 100.6 17.2 71.2 11.1 AD-901429.1 94.4 7.7 118.8 28.5 93.2 6.7 101.2 12.0 92.5 7.3 64.3 18.4 85.1 6.6 75.3 16.0 AD-901402.1 99.4 13.8 118.9 25.5 107.3 6.3 100.5 9.5 99.8 15.7 108.9 8.5 94.6 9.5 75.2 18.7 AD-901430.1 95.7 16.4 119.8 30.7 82.8 5.5 98.2 12.4 95.2 10.9 90.0 30.2 72.8 6.4 74.4 9.7 AD-901369.1 79.4 12.7 135.3 57.4 89.5 2.4 75.0 10.6 112.5 21.8 82.9 15.8 88.4 2.8 66.5 21.4

TABLE 6B VEGF-A endogenous in vitro multi-dose screen with one set of exemplary human VEGF-A siRNAs ARPE-19 hTERT RPE-1 50 10 1 0.1 50 10 1 0.1 Sample Name nM StDev nM StDev nM StDev nM StDev nM StDev nM StDev nM StDev nM StDev AD-953340.1 18.6 2.1 2.7 0.7 28.5 3.4 106.5 28.3 40.1 3.9 25.2 10.0 38.7 7.5 52.2 15.2 AD-953336.1 25.7 1.4 3.7 0.9 26.5 0.5 159.2 61.6 45.5 5.2 26.3 8.1 41.0 8.9 68.2 8.3 AD-953363.1 17.3 2.9 3.9 0.9 33.5 6.0 57.0 7.1 37.2 6.8 24.6 5.6 38.1 5.9 39.4 8.0 AD-953338.1 23.1 2.7 4.8 1.8 41.2 8.4 92.1 21.1 54.6 7.9 35.3 7.5 51.1 1.4 76.1 10.8 AD-953367.1 25.6 1.6 5.1 0.6 39.3 6.9 55.8 5.1 62.2 8.0 33.2 10.3 61.2 8.6 49.4 7.6 AD-953337.1 29.5 4.0 5.6 3.4 28.2 2.0 129.8 30.5 45.4 8.6 23.8 4.5 44.8 3.6 60.6 8.0 AD-953342.1 18.9 2.3 5.6 3.6 31.6 4.9 149.8 56.1 35.2 4.0 28.4 6.6 39.0 2.7 73.7 11.9 AD-953350.1 30.0 3.6 5.8 4.1 46.6 8.3 78.3 22.9 70.5 10.7 42.6 14.8 49.3 14.2 85.8 3.8 AD-953352.1 28.5 2.8 6.1 0.5 41.5 5.7 113.4 13.2 52.8 5.8 40.1 9.3 53.0 7.6 72.9 7.3 AD-953368.1 33.3 1.6 6.2 1.0 35.2 2.3 48.2 3.6 58.8 5.7 39.5 11.2 50.1 7.2 50.5 2.2 AD-953344.1 17.8 1.6 6.5 1.9 38.5 5.7 66.5 11.3 29.5 4.0 28.9 7.1 49.6 7.0 75.8 12.9 AD-953339.1 26.5 4.9 6.7 5.5 34.4 6.2 120.0 30.4 41.7 3.6 26.2 3.7 36.0 9.0 59.1 7.0 AD-953387.1 20.6 1.0 6.9 0.3 40.9 5.4 64.9 6.1 21.0 2.8 31.3 5.4 69.7 15.0 57.4 7.3 AD-953375.1 37.1 2.8 7.4 1.1 48.8 6.5 73.4 3.9 90.7 19.7 62.5 8.1 72.2 11.9 64.3 16.3 AD-953355.1 42.7 5.3 7.5 0.7 62.2 2.4 137.9 24.5 43.9 5.5 40.6 8.0 52.6 10.0 67.1 5.5 AD-953341.1 20.2 2.9 7.7 4.5 22.3 3.3 106.6 21.7 55.5 5.5 34.7 7.7 32.2 8.3 68.4 5.8 AD-953370.1 36.8 3.0 7.8 1.9 55.5 7.5 72.3 6.0 38.0 7.8 26.9 4.7 63.9 12.6 51.9 6.9 AD-953362.1 57.6 4.3 8.2 1.1 54.3 8.3 76.8 3.6 108.9 11.3 48.1 10.1 63.0 11.0 52.1 10.4 AD-953322.1 59.6 3.6 8.2 1.3 61.4 4.5 105.4 14.2 103.5 9.6 55.4 15.9 66.5 7.4 78.4 11.5 AD-953332.1 59.2 3.3 8.3 1.3 58.3 3.8 106.7 20.6 95.1 15.8 55.0 9.1 74.5 1.5 66.2 8.9 AD-953371.1 41.1 3.2 8.5 2.6 49.1 7.2 64.1 10.0 75.1 16.0 51.2 18.1 60.0 8.7 53.0 11.6 AD-953331.1 68.0 1.2 8.8 1.7 60.3 5.5 92.6 24.7 151.3 18.2 88.8 33.3 85.2 21.0 87.4 5.6 AD-953323.1 66.8 5.0 9.1 1.7 64.2 2.1 109.8 19.9 93.6 2.2 53.0 7.3 74.7 4.9 77.0 11.6 AD-953351.1 21.2 2.3 9.2 9.5 39.8 6.9 74.4 8.0 49.7 3.2 26.0 2.5 41.2 11.0 63.6 8.2 AD-953386.1 31.8 1.6 9.3 1.0 65.3 6.3 87.4 5.1 38.7 0.4 50.4 11.0 82.9 17.7 67.9 13.0 AD-953394.1 35.4 2.0 9.5 5.1 50.8 5.3 72.7 3.3 66.5 16.5 49.7 10.5 77.2 14.7 57.8 21.7 AD-953359.1 36.4 3.8 9.7 2.9 46.9 6.3 72.9 7.4 74.8 9.6 46.2 6.0 63.9 10.8 50.7 7.9 AD-953329.1 68.0 10.2 9.7 1.7 59.7 1.6 100.3 22.8 94.3 7.1 56.9 16.4 76.7 10.0 79.8 9.0 AD-953361.1 45.2 2.9 10.4 8.6 53.6 4.8 68.4 5.4 78.6 10.9 50.2 11.3 68.6 9.3 47.8 9.6 AD-953319.1 100.2 2.8 10.6 1.4 71.5 10.3 161.5 76.4 189.6 19.4 69.0 22.5 84.3 4.7 93.2 18.2 AD-953360.1 96.2 10.8 10.7 1.5 49.6 5.2 76.7 6.2 140.9 16.5 69.1 20.5 82.4 9.0 54.9 2.8 AD-953324.1 63.0 2.8 11.2 4.8 53.8 4.5 96.6 27.2 131.1 18.6 67.5 16.9 86.3 18.2 75.5 18.1 AD-953378.1 57.9 8.3 11.5 2.3 72.2 7.6 92.4 6.2 47.1 10.1 55.3 8.2 85.7 17.9 60.5 14.4 AD-953369.1 43.6 4.7 11.8 6.0 64.4 10.3 79.8 5.1 68.6 9.7 56.3 9.1 70.2 5.4 58.7 14.7 AD-953347.1 77.6 5.1 11.9 3.7 63.3 7.7 66.5 16.2 112.3 25.0 62.1 13.1 62.8 13.8 76.4 6.3 AD-953365.1 28.7 4.1 12.3 3.5 35.7 4.9 61.7 7.9 36.6 7.3 23.6 5.0 56.5 3.9 50.9 4.6 AD-953374.1 10.7 0.3 12.3 18.3 35.8 27.4 33.8 7.4 30.0 3.5 33.2 4.8 39.2 4.0 46.2 7.9 AD-953384.1 57.1 2.9 12.9 4.8 76.3 9.0 82.4 5.1 86.2 5.2 67.4 3.8 105.9 23.9 66.1 13.2 AD-953376.1 57.8 2.7 12.9 2.0 81.7 9.3 94.3 1.0 69.2 14.7 73.1 23.1 96.7 8.4 70.3 10.8 AD-953354.1 49.1 5.5 13.5 5.6 67.9 5.6 141.1 30.9 40.5 6.7 42.2 10.8 57.0 6.1 78.8 9.6 AD-953385.1 65.0 3.9 13.5 2.0 79.8 8.9 97.1 13.1 115.8 16.5 107.5 18.6 124.6 8.8 71.9 7.1 AD-953346.1 38.0 3.2 14.1 11.9 56.3 4.7 68.3 13.8 58.6 8.1 42.7 7.4 53.9 11.6 75.2 5.3 AD-953366.1 28.1 3.0 14.3 18.9 47.6 4.2 72.4 7.9 47.0 5.3 32.5 6.0 40.4 8.8 61.7 14.1 AD-953382.1 67.1 5.3 14.6 3.6 80.8 7.7 99.5 6.7 143.5 30.8 67.6 18.2 91.3 11.9 67.0 12.6 AD-953320.1 65.2 3.4 14.7 6.0 69.8 9.1 139.3 11.6 121.6 7.8 55.2 11.1 97.9 7.0 92.3 10.4 AD-953379.1 64.5 8.5 17.0 5.4 76.3 3.5 85.5 6.7 60.1 12.9 68.0 26.1 85.4 3.5 49.6 12.0 AD-953321.1 66.2 5.0 17.1 13.7 58.7 5.7 125.7 34.8 111.0 19.6 59.2 14.5 72.7 12.4 80.3 11.7 AD-953377.1 55.4 3.9 17.2 8.0 75.0 6.1 83.8 4.1 54.9 8.5 57.6 9.1 82.9 6.1 70.7 12.3 AD-953392.1 88.8 1.4 17.5 2.0 90.9 10.8 96.0 3.6 125.6 16.1 102.0 20.4 117.0 26.2 88.2 14.3 AD-953373.1 35.7 3.9 17.7 12.7 41.4 3.3 61.2 9.4 56.7 9.0 50.8 5.4 66.6 5.1 70.4 14.3 AD-953364.1 22.5 1.9 18.1 6.4 35.7 2.4 53.4 3.8 21.2 2.6 20.1 5.9 30.5 4.3 45.0 7.9 AD-953330.1 56.3 6.5 18.2 8.1 54.0 2.8 97.5 23.3 109.0 14.5 69.9 11.8 68.7 8.6 74.6 9.6 AD-953353.1 30.9 2.4 19.3 14.9 51.1 3.9 145.6 33.7 59.1 11.7 45.4 10.7 57.3 8.9 80.5 6.8 AD-953343.1 28.4 1.6 19.4 24.7 31.5 2.1 50.2 12.1 51.1 7.7 41.1 4.8 50.6 3.0 76.9 6.2 AD-953390.1 82.4 4.8 19.8 2.3 92.7 10.4 103.4 7.6 132.1 19.0 96.0 20.4 114.6 24.4 80.3 14.4 AD-953345.1 26.9 4.4 20.4 16.6 51.9 6.3 91.9 13.0 35.7 10.4 33.1 6.6 37.6 9.9 65.9 6.8 AD-953358.1 30.5 1.7 20.6 17.0 40.7 6.0 66.2 3.7 67.1 7.3 46.2 13.7 62.8 16.0 52.4 10.8 AD-953383.1 58.5 4.0 21.4 9.2 65.7 5.7 83.3 3.1 70.5 9.4 65.2 10.6 82.7 14.0 80.7 8.9 AD-953372.1 34.3 4.8 24.0 7.7 45.9 5.8 65.7 4.7 52.6 5.2 42.1 13.1 65.9 9.1 54.7 6.6 AD-953328.1 54.5 3.9 24.1 19.0 60.6 5.7 112.1 3.8 137.7 50.8 66.1 5.8 82.7 13.1 79.1 7.2 AD-953393.1 42.8 3.6 25.9 33.0 63.6 6.1 87.9 3.4 49.7 9.2 56.8 6.9 81.0 6.8 74.0 14.3 AD-953307.1 39.3 2.0 26.1 2.3 50.3 3.0 144.4 30.6 49.9 4.0 34.1 7.3 65.1 4.0 56.8 9.9 AD-953308.1 36.9 6.6 26.3 1.3 50.9 3.7 136.2 32.8 30.3 7.1 24.2 4.4 53.4 4.8 42.3 8.9 AD-953327.1 63.7 9.2 27.4 29.5 52.5 1.5 108.8 24.5 85.5 42.1 48.8 10.7 72.2 10.9 82.3 7.2 AD-953335.1 77.1 5.8 28.3 19.0 53.0 4.4 115.1 32.1 144.1 17.3 72.8 17.2 84.8 11.6 95.4 8.0 AD-953414.1 21.5 1.7 28.3 1.6 41.8 4.6 61.5 8.3 40.1 2.5 31.3 7.9 49.3 12.3 66.0 17.8 AD-953412.1 16.3 0.7 28.4 3.6 33.1 5.3 65.1 4.0 37.5 11.0 35.3 3.6 35.8 9.0 70.7 14.2 AD-953411.1 15.4 2.0 29.5 3.7 42.7 5.0 65.5 6.0 30.9 9.0 26.2 4.3 48.9 7.9 57.2 13.3 AD-953410.1 16.4 1.2 29.6 2.0 44.7 3.6 68.0 2.7 26.2 3.8 26.4 7.0 49.8 9.2 55.9 17.7 AD-953408.1 21.0 1.4 29.7 2.3 38.4 4.8 72.1 6.4 33.1 4.7 35.5 2.9 60.9 14.6 80.9 23.6 AD-953326.1 53.8 7.2 30.2 17.6 50.4 3.4 115.3 25.4 115.2 12.1 59.7 19.5 77.5 18.1 83.0 11.6 AD-953300.1 49.4 5.2 30.6 1.7 52.2 5.5 176.7 31.5 51.7 16.4 44.1 6.6 65.9 17.3 39.7 7.3 AD-953389.1 90.9 7.3 31.6 16.2 93.6 8.0 99.4 7.4 144.1 18.2 126.7 8.2 114.4 15.0 93.8 22.2 AD-953415.1 23.3 1.8 32.0 1.8 41.3 3.0 62.8 8.8 65.9 6.6 52.5 5.9 53.3 18.2 70.5 19.8 AD-953309.1 42.1 1.6 32.3 3.0 69.9 2.0 148.4 9.6 40.6 7.2 42.0 7.1 91.4 19.0 48.5 11.7 AD-953391.1 78.3 7.4 32.9 32.3 86.1 7.2 95.6 4.9 108.7 4.5 93.3 10.9 119.0 24.5 87.2 15.4 AD-953395.1 53.9 5.2 33.3 21.8 70.2 7.5 87.9 3.9 42.9 5.1 49.4 8.8 83.2 6.3 62.2 11.3 AD-953303.1 48.8 2.8 34.0 9.2 44.4 3.8 133.6 33.0 69.9 7.6 42.4 3.8 62.7 7.0 54.4 12.6 AD-953405.1 37.6 9.8 34.2 5.3 42.1 8.8 60.5 3.6 58.3 17.4 37.6 7.0 59.1 16.6 78.3 6.9 AD-953305.1 52.0 6.9 34.8 1.6 56.5 5.1 108.4 23.2 73.0 10.3 41.9 6.0 68.4 9.8 69.3 6.9 AD-953380.1 63.5 8.0 35.1 28.6 72.8 5.2 84.6 3.4 54.0 2.4 49.6 9.1 82.2 16.6 54.1 9.0 AD-953349.1 62.9 2.8 35.3 4.1 61.4 6.2 71.3 23.0 106.7 20.9 64.7 14.2 67.5 26.3 92.3 7.5 AD-953381.1 66.3 2.9 35.5 22.1 83.5 7.5 99.8 6.3 80.8 10.0 67.8 8.9 99.2 18.4 76.9 21.1 AD-953318.1 73.1 4.0 37.6 32.2 63.4 2.6 198.1 91.7 132.1 16.9 66.1 30.7 81.7 17.2 88.7 3.1 AD-953348.1 46.6 2.7 38.4 9.6 52.5 5.4 136.3 31.4 46.5 14.1 38.9 9.4 48.8 9.2 74.5 10.8 AD-953409.1 24.1 1.4 39.4 2.8 46.7 7.0 76.5 7.7 32.7 3.5 28.5 4.8 59.1 9.8 65.9 21.4 AD-953306.1 69.9 8.3 39.7 4.7 62.4 5.2 134.9 32.7 127.5 9.5 69.9 20.8 84.4 4.7 44.9 5.0 AD-953316.1 59.6 4.5 40.3 3.9 54.4 3.5 94.5 8.4 129.3 20.3 58.5 9.4 72.2 12.8 58.6 15.8 AD-953325.1 69.2 6.7 41.8 24.8 68.9 7.7 153.7 53.1 112.9 25.0 66.6 11.7 76.9 13.0 70.1 9.9 AD-953299.1 64.9 6.3 42.3 2.6 70.3 5.0 164.5 59.7 67.8 20.4 65.1 16.9 83.0 9.2 50.0 7.5 AD-953416.1 29.1 3.7 42.4 3.1 41.8 3.8 53.8 4.6 79.4 10.1 51.0 9.1 57.1 5.1 63.4 7.3 AD-953315.1 62.1 5.3 42.6 1.1 55.8 2.8 95.3 27.0 134.9 23.0 84.8 6.7 73.0 6.5 58.6 17.0 AD-953314.1 55.4 5.5 42.6 2.8 55.8 3.3 115.4 15.1 106.7 3.2 62.9 8.0 81.7 8.5 53.3 21.1 AD-953298.1 65.3 4.3 44.0 2.2 67.5 6.6 165.9 13.3 60.5 6.8 46.5 9.6 89.0 8.6 57.6 7.6 AD-953406.1 27.2 1.4 44.5 5.0 58.8 5.8 84.3 2.0 50.0 4.6 37.8 4.4 74.7 8.6 75.2 29.3 AD-953399.1 32.1 2.3 45.5 1.4 44.3 3.6 64.6 2.8 84.3 9.4 43.2 8.2 80.6 5.7 79.7 20.7 AD-953333.1 54.7 4.9 45.7 17.9 63.1 3.7 139.5 14.5 104.8 10.4 50.6 15.6 65.9 13.4 66.1 14.1 AD-953313.1 57.3 3.7 45.9 2.5 64.6 2.4 107.6 22.5 81.2 19.1 61.7 8.4 89.7 4.0 83.2 4.5 AD-953302.1 49.5 2.8 47.0 5.0 61.7 3.9 108.3 31.0 41.6 11.9 48.7 7.2 57.5 8.9 68.8 4.8 AD-953317.1 76.9 5.3 50.0 3.1 72.6 9.3 145.3 34.8 77.9 18.1 60.9 12.4 71.3 9.7 50.4 14.7 AD-953357.1 61.0 5.5 50.3 33.1 70.6 8.6 94.4 5.1 70.4 16.9 59.5 19.7 99.6 27.9 52.4 8.5 AD-953301.1 62.2 7.8 50.5 5.6 82.8 5.7 193.0 33.0 47.3 16.7 38.2 3.8 63.5 8.8 53.8 10.8 AD-953304.1 60.9 6.6 51.4 2.2 75.4 1.9 133.7 36.1 133.7 5.8 89.6 12.1 95.5 8.0 65.9 8.4 AD-953297.1 77.5 3.2 52.2 4.2 74.9 4.8 142.3 32.5 118.2 19.7 85.8 5.2 99.1 11.6 59.7 8.0 AD-953388.1 80.1 5.6 53.7 40.5 91.0 9.7 95.9 10.1 139.5 8.4 89.7 6.8 107.9 17.8 98.0 21.3 AD-953407.1 37.2 4.3 53.9 9.4 47.8 6.2 72.1 4.3 61.2 7.6 43.4 16.4 66.1 17.3 74.5 6.9 AD-953397.1 56.1 5.0 57.1 3.8 58.1 9.9 77.8 5.1 96.4 13.5 52.9 9.6 94.9 13.8 76.0 9.4 AD-953398.1 47.8 2.1 57.4 6.6 51.1 5.3 71.6 2.3 62.1 7.0 51.9 7.5 99.5 13.7 93.8 13.7 AD-953396.1 52.3 7.4 59.5 5.6 63.4 6.2 86.9 5.8 85.3 10.0 52.7 5.7 98.7 8.2 101.8 11.6 AD-953356.1 80.6 1.8 63.1 11.3 82.1 13.5 119.0 27.2 93.5 11.4 65.8 9.2 76.8 20.8 61.8 12.3 AD-953422.1 47.6 4.2 63.3 7.6 189.1 56.5 87.2 3.9 56.5 5.7 58.9 8.6 36.4 2.3 74.3 18.0 AD-953413.1 56.5 4.9 64.0 8.2 68.9 4.5 95.5 9.7 78.1 8.7 72.4 12.0 81.1 9.4 90.4 15.6 AD-953294.1 97.1 15.2 64.2 4.2 77.3 15.5 96.7 15.6 96.8 35.0 71.6 5.9 76.3 3.3 69.9 12.0 AD-953421.1 53.2 6.7 65.0 2.9 197.8 29.8 101.0 7.9 102.0 13.5 65.6 7.9 49.4 12.2 94.1 8.4 AD-953310.1 117.3 11.7 66.7 16.2 93.9 3.2 148.4 45.0 149.3 31.4 91.4 14.3 97.9 3.5 72.3 17.0 AD-953296.1 103.9 2.9 67.6 5.3 85.2 8.1 137.2 17.9 187.5 42.8 110.2 4.6 116.2 10.5 56.8 9.4 AD-953402.1 55.1 2.2 67.6 4.0 63.8 6.1 84.1 7.6 83.2 19.9 75.6 8.7 89.0 23.0 77.5 11.6 AD-953312.1 86.0 14.3 71.1 4.1 74.3 3.3 145.1 57.4 171.9 22.0 84.6 5.5 100.5 13.7 94.5 9.6 AD-953295.1 94.5 4.7 71.6 3.7 83.7 11.4 136.9 20.4 122.9 21.2 90.4 14.2 85.2 26.5 64.3 18.0 AD-953420.1 58.3 2.3 72.8 7.3 177.1 37.4 91.6 3.5 74.6 3.6 60.0 6.7 52.2 7.9 96.5 11.0 AD-953423.1 47.1 3.4 73.0 14.3 215.7 13.2 93.0 6.7 90.7 6.7 64.9 9.2 43.6 7.2 89.1 15.2 AD-953403.1 62.9 12.6 73.9 5.6 58.5 5.9 77.8 6.5 102.6 12.5 62.1 6.0 96.2 14.6 75.4 7.3 AD-953400.1 52.1 2.7 74.8 10.9 58.9 4.4 78.3 6.7 83.2 11.5 48.5 14.9 86.8 26.5 84.6 8.4 AD-953404.1 73.3 4.0 76.2 8.8 72.0 3.4 92.1 3.5 133.1 19.8 70.7 8.1 103.8 14.5 97.1 12.4 AD-953334.1 59.9 3.8 76.5 27.3 57.6 1.4 115.3 6.2 178.1 16.6 89.4 25.1 78.2 17.1 98.1 8.5 AD-953418.1 57.2 3.7 79.6 12.5 81.6 6.1 92.3 4.1 100.0 20.4 57.2 11.1 70.4 11.1 79.1 4.1 AD-953401.1 61.5 3.7 81.6 4.1 78.6 9.2 96.9 10.8 93.0 8.4 67.9 27.9 105.6 5.0 84.0 6.1 AD-953417.1 57.8 1.8 82.5 1.5 84.7 11.1 89.1 6.1 86.4 8.4 65.7 14.4 69.9 7.2 82.8 8.5 AD-953311.1 113.2 5.8 91.3 6.1 93.9 8.2 108.7 25.4 156.0 13.3 86.4 10.1 92.6 5.7 77.0 15.2 AD-953419.1 74.7 3.6 94.8 6.4 93.2 5.5 92.2 5.3 85.9 13.4 57.3 8.4 84.5 16.8 76.8 7.9

TABLE 6C VEGF-A endogenous in vitro multi-dose screen with one set of exemplary human VEGF-A siRNAs ARPE-19 hTERT RPE-1 50 10 1 0.1 50 10 1 0.1 Sample Name nM StDev nM StDev nM StDev nM StDev nM StDev nM StDev nM StDev nM StDev AD-953504.1 17.3 0.6 19.8 5.4 48.7 32.4 56.5 9.3 19.9 3.2 22.2 2.8 61.7 21.8 65.7 15.9 AD-953481.1 33.7 2.2 28.1 2.4 60.4 3.8 72.8 1.5 42.5 9.2 25.3 3.5 68.8 28.1 78.2 28.1 AD-953472.1 31.3 1.6 30.1 1.2 54.1 4.4 80.0 6.6 45.9 7.1 30.6 2.9 64.9 14.9 91.0 18.4 AD-953517.1 29.5 3.2 30.3 1.4 45.6 5.1 74.4 4.9 37.6 4.9 36.3 7.9 69.4 18.5 74.5 10.3 AD-953471.1 33.4 0.9 30.5 2.2 47.1 5.1 87.8 15.3 39.7 11.2 28.0 5.0 68.2 12.1 77.6 14.1 AD-953493.1 46.0 10.0 33.3 4.0 64.9 9.0 81.1 4.1 24.9 5.5 22.5 2.5 50.8 23.2 54.0 12.0 AD-953498.1 46.8 3.8 34.5 1.5 55.9 5.9 76.7 8.9 43.1 2.2 31.2 4.9 64.8 16.8 78.0 19.6 AD-953467.1 42.3 6.8 34.6 3.5 59.8 14.0 78.3 11.6 42.8 3.2 31.1 8.9 56.9 9.0 94.4 12.2 AD-953545.1 31.2 0.8 35.4 8.1 48.5 6.1 73.7 7.0 55.3 8.9 42.0 8.6 85.9 16.6 79.4 7.1 AD-953466.1 53.7 2.9 36.9 3.0 61.5 20.5 74.1 8.9 62.8 5.4 39.6 3.4 65.3 7.7 103.3 34.2 AD-953494.1 49.2 2.9 38.4 1.6 66.3 7.1 85.6 4.5 32.7 3.4 26.5 8.0 46.1 7.1 45.7 6.8 AD-953470.1 66.5 2.0 40.5 3.4 63.8 13.5 74.9 5.0 56.4 9.5 34.8 7.3 64.6 9.9 105.6 20.0 AD-953473.1 59.2 2.9 42.3 5.7 61.7 4.1 80.9 4.5 72.8 11.9 44.2 3.0 77.6 25.2 110.0 27.1 AD-953474.1 61.5 7.9 42.4 1.6 72.9 7.5 86.4 5.6 63.8 4.5 48.8 2.4 85.5 29.3 107.1 18.8 AD-953480.1 41.7 3.9 43.3 1.9 72.9 5.0 91.6 11.4 47.1 9.2 47.2 16.2 77.8 38.4 93.8 27.0 AD-953503.1 56.4 2.3 43.9 4.9 59.9 7.6 88.7 10.2 44.2 13.0 57.5 9.7 90.0 9.8 83.2 11.7 AD-953478.1 55.1 8.7 44.3 4.1 64.1 4.5 83.3 8.7 38.9 3.2 52.3 22.9 70.3 23.4 78.1 9.5 AD-953540.1 29.4 2.2 44.6 10.3 60.9 5.7 94.9 10.3 23.4 6.6 22.7 7.9 59.7 12.0 81.5 3.3 AD-953500.1 46.3 10.0 45.8 4.0 70.6 10.0 91.2 4.6 30.4 4.6 31.2 1.2 79.7 16.0 80.1 10.5 AD-953476.1 45.9 6.5 46.7 2.3 78.7 3.7 91.8 4.7 40.4 4.4 44.7 10.0 84.2 14.5 118.2 21.3 AD-953492.1 61.2 1.9 47.1 3.8 71.8 6.2 85.2 8.4 58.2 7.0 44.7 4.1 79.6 20.6 73.1 13.5 AD-953495.1 60.7 3.5 47.4 3.9 64.3 15.3 84.0 5.7 54.3 7.7 39.5 8.5 69.7 9.1 68.8 26.1 AD-953497.1 55.5 1.6 47.9 5.1 65.9 9.6 89.9 8.5 61.5 11.8 45.8 11.0 81.4 21.9 86.5 11.4 AD-953535.1 52.1 10.8 48.3 10.8 56.0 5.5 76.1 4.9 52.6 8.9 42.6 8.1 94.1 22.4 100.5 4.2 AD-953505.1 60.3 6.1 48.4 2.0 64.4 2.4 94.7 10.5 54.9 8.8 59.1 7.3 104.2 19.2 88.3 14.3 AD-953524.1 57.5 1.2 48.9 2.1 50.9 20.8 93.7 6.7 44.2 4.8 51.2 8.8 68.3 25.3 64.5 14.1 AD-953475.1 55.7 2.1 49.6 3.8 69.2 7.9 83.9 5.0 54.2 11.0 41.8 8.6 87.2 24.9 105.6 23.8 AD-953491.1 68.9 9.7 49.9 4.6 69.4 2.7 92.7 10.9 54.5 8.4 54.9 20.8 72.8 15.0 73.5 16.3 AD-953436.1 96.6 1.6 50.6 2.1 63.1 13.9 80.4 4.8 72.6 8.9 72.9 2.2 108.8 9.8 67.6 24.6 AD-953502.1 62.8 9.4 50.8 3.8 74.9 6.4 87.0 13.4 52.2 4.0 42.0 8.7 75.8 17.7 49.4 4.5 AD-953461.1 61.7 5.8 51.1 5.0 74.3 17.8 74.6 2.7 66.9 10.6 62.6 8.7 78.9 3.2 94.0 19.6 AD-953544.1 42.2 3.8 51.3 8.4 61.5 6.6 89.4 2.2 47.4 9.0 45.8 10.0 83.1 19.6 92.4 16.0 AD-953462.1 64.6 3.0 51.5 5.9 84.5 16.8 80.3 6.5 53.0 14.9 48.2 11.8 84.0 18.2 114.8 20.3 AD-953496.1 52.0 5.9 51.6 5.0 79.2 5.6 89.8 6.5 55.1 5.5 58.3 4.8 97.3 22.7 83.9 25.2 AD-953516.1 59.7 7.4 51.9 2.3 71.2 11.2 93.4 13.2 49.8 14.2 43.5 13.2 86.5 9.4 78.4 11.1 AD-953483.1 61.6 4.6 52.0 2.3 69.5 17.6 92.7 9.0 52.2 7.3 47.3 13.2 99.1 21.9 94.1 22.9 AD-953499.1 64.5 8.2 52.7 3.2 71.1 11.7 105.8 6.7 70.7 10.1 46.6 5.0 90.6 15.3 85.6 14.6 AD-953541.1 48.8 10.7 53.1 12.9 55.9 6.0 90.1 9.3 32.7 6.8 31.2 3.2 68.9 24.2 79.9 10.0 AD-953538.1 38.4 5.2 54.1 10.1 68.1 9.3 99.0 6.5 32.1 8.8 29.3 3.2 89.4 17.2 88.1 7.9 AD-953430.1 85.4 7.5 54.7 2.9 90.8 20.1 82.8 2.9 57.6 4.4 55.1 11.1 80.2 14.7 65.6 13.5 AD-953485.1 66.8 2.1 54.9 3.8 84.8 6.5 90.0 2.2 39.0 8.2 42.3 5.6 66.0 8.7 95.6 22.6 AD-953468.1 65.7 3.5 55.1 2.6 86.4 11.6 97.3 11.9 50.2 8.8 41.0 3.6 88.9 21.6 108.5 25.1 AD-953444.1 63.2 2.2 55.3 3.8 81.6 15.3 87.3 9.0 55.0 5.1 76.5 12.6 108.8 22.7 70.4 18.1 AD-953460.1 65.6 10.0 55.6 3.2 85.2 10.4 83.1 6.6 56.8 15.0 46.3 6.9 87.1 6.8 121.0 12.9 AD-953539.1 67.0 8.5 56.1 11.5 62.2 2.9 97.3 4.3 47.1 5.3 30.5 6.1 65.5 15.9 89.6 10.6 AD-953484.1 60.1 16.2 56.6 5.9 86.0 4.8 94.4 6.4 41.3 9.9 46.9 10.8 81.1 26.2 97.8 20.4 AD-953457.1 69.0 26.0 57.8 5.5 79.7 22.5 78.0 8.4 96.5 61.4 42.8 3.1 85.0 11.8 84.4 28.0 AD-953459.1 67.3 9.8 57.9 4.0 82.0 15.1 85.5 2.9 66.1 9.8 52.4 5.4 90.4 4.6 111.4 7.6 AD-953437.1 74.6 5.0 58.6 4.4 87.7 20.7 84.6 6.4 53.5 5.2 70.2 20.0 89.8 7.7 77.7 21.8 AD-953458.1 70.7 8.8 59.3 2.4 81.8 21.2 79.2 5.7 75.8 13.1 47.7 2.1 97.0 12.3 107.7 31.1 AD-953453.1 69.9 3.5 59.5 4.3 74.0 17.2 84.4 11.6 61.1 9.9 59.0 5.7 89.6 5.5 88.0 27.3 AD-953428.1 67.5 3.0 60.6 2.3 92.1 20.1 86.2 0.7 36.2 5.6 59.5 10.0 108.5 13.2 70.4 11.3 AD-953501.1 77.3 7.8 60.9 5.8 91.7 5.6 100.8 7.2 67.9 9.6 61.3 14.9 80.5 24.9 73.2 5.7 AD-953482.1 76.7 1.5 61.5 3.4 94.6 8.4 100.1 5.4 71.8 9.0 53.7 8.6 115.9 9.7 101.9 20.8 AD-953446.1 94.0 4.3 61.6 5.8 76.4 15.8 75.6 5.7 64.1 4.6 74.9 8.7 96.8 19.8 64.5 10.4 AD-953488.1 65.7 3.3 61.8 3.9 81.2 15.8 95.9 9.8 81.3 5.3 60.2 18.5 123.3 46.3 76.4 23.7 AD-953434.1 58.9 3.9 61.9 3.1 91.4 17.9 94.1 10.6 49.7 5.3 92.6 15.2 123.1 12.0 71.5 10.1 AD-953546.1 49.9 1.9 63.3 14.5 59.7 4.4 80.8 2.1 48.2 2.7 41.6 12.9 72.9 5.4 72.9 8.7 AD-953529.1 59.0 3.6 64.8 9.9 63.9 2.4 78.3 1.9 61.5 5.7 48.5 7.4 117.3 20.3 91.1 15.1 AD-953433.1 95.2 10.7 64.8 6.3 78.5 16.2 82.5 2.6 69.0 5.2 109.4 7.2 107.4 13.2 98.9 35.0 AD-953456.1 68.1 8.4 64.9 4.7 95.7 22.5 94.1 2.7 86.8 14.7 56.3 7.0 89.1 10.7 89.1 12.9 AD-953435.1 69.8 3.4 65.1 4.1 88.1 18.6 92.9 4.8 57.5 9.2 75.3 5.7 104.3 2.4 71.9 13.2 AD-953438.1 70.2 4.8 66.4 5.8 105.5 19.6 88.2 3.9 46.0 7.7 61.7 19.7 90.1 14.9 71.7 16.9 AD-953452.1 80.0 5.5 66.9 6.7 96.6 21.5 89.5 10.8 53.2 3.6 46.9 7.7 94.3 14.7 104.6 25.8 AD-953489.1 86.9 6.5 69.3 5.5 89.1 10.0 97.0 8.7 82.0 16.1 80.6 15.6 125.9 15.7 77.4 19.4 AD-953445.1 104.1 7.0 71.5 3.4 88.6 21.2 85.2 10.1 68.1 3.0 91.0 8.8 103.1 9.2 80.5 32.1 AD-953432.1 62.6 4.1 71.6 4.0 106.7 23.0 109.1 13.6 38.2 5.3 69.3 6.0 104.2 19.0 88.8 34.4 AD-953509.1 96.2 8.8 71.8 2.2 68.5 36.5 93.1 7.2 71.0 33.3 70.4 7.3 77.7 11.3 65.4 12.1 AD-953490.1 89.4 6.4 72.2 2.5 85.2 6.8 95.5 8.7 109.5 9.8 68.7 8.2 106.9 14.9 74.4 6.7 AD-953448.1 67.8 3.6 72.7 5.1 101.5 20.3 94.8 9.3 76.1 25.3 61.8 6.4 110.6 35.5 79.5 29.6 AD-953450.1 78.6 2.3 72.8 5.4 96.9 14.3 91.0 7.6 72.1 7.7 53.3 4.3 120.8 11.3 117.2 41.1 AD-953443.1 96.4 8.9 73.3 2.2 96.4 22.9 90.7 4.5 60.8 3.2 100.7 16.4 95.8 18.2 83.7 14.5 AD-953525.1 81.5 6.2 73.4 4.5 79.8 16.8 90.4 2.8 53.0 4.5 69.3 8.5 86.0 16.0 90.0 8.6 AD-953523.1 74.3 4.9 73.4 8.8 88.1 6.8 100.9 5.1 58.3 15.3 91.1 6.6 116.2 14.1 78.2 3.5 AD-953507.1 78.1 8.2 73.7 5.3 86.3 6.8 98.8 5.4 51.1 14.2 64.7 6.5 104.2 32.5 75.3 23.7 AD-953451.1 87.6 7.6 74.0 8.2 92.4 16.6 90.3 3.1 95.8 23.0 56.1 5.2 112.4 8.5 110.3 6.9 AD-953429.1 91.2 8.9 74.2 4.9 99.8 24.4 93.0 7.0 57.0 9.1 66.7 10.2 99.6 11.7 79.4 16.7 AD-953469.1 84.5 11.2 74.6 5.1 109.2 18.8 95.6 5.9 75.1 4.5 62.7 8.0 88.1 7.8 117.5 17.4 AD-953463.1 88.7 17.1 74.8 4.8 101.9 20.8 99.2 5.1 70.1 8.6 75.6 20.8 80.2 25.5 88.4 11.1 AD-953454.1 106.1 7.0 74.9 6.7 95.6 26.8 86.4 9.1 76.3 20.1 67.7 21.9 95.9 14.3 84.0 35.8 AD-953455.1 99.0 14.1 75.0 5.4 101.7 25.0 98.1 5.5 81.5 17.4 72.9 19.3 76.3 14.6 69.1 15.5 AD-953511.1 83.7 3.8 75.3 6.2 86.7 5.4 100.7 8.8 76.0 31.8 71.6 10.7 131.4 22.1 94.7 13.1 AD-953447.1 95.2 8.5 75.6 4.2 101.4 18.3 89.6 4.2 67.5 9.6 73.0 10.4 75.9 14.6 76.1 16.4 AD-953424.1 78.8 3.7 75.8 8.2 96.4 26.2 99.3 5.8 56.2 9.0 98.3 31.6 112.0 18.9 68.1 16.5 AD-953506.1 82.9 3.4 76.4 3.9 94.6 10.2 98.2 7.0 54.4 7.9 80.4 12.3 104.5 27.0 85.1 2.6 AD-953537.1 71.6 3.2 76.4 16.4 81.7 10.1 93.7 6.6 61.5 11.6 52.7 14.9 104.7 9.9 102.3 8.2 AD-953477.1 104.2 11.7 76.5 4.8 93.7 6.9 90.6 5.6 77.5 20.8 62.6 12.8 75.1 22.1 105.4 29.2 AD-953479.1 90.5 8.9 77.0 10.7 99.9 16.6 109.0 9.6 99.6 9.7 74.8 9.6 116.9 25.5 104.0 21.8 AD-953439.1 102.3 7.6 77.2 4.5 100.8 26.6 97.8 5.7 68.0 8.2 80.5 24.1 72.4 18.5 66.2 17.0 AD-953431.1 86.4 7.7 77.5 7.5 116.2 20.7 93.5 15.3 52.4 2.4 45.7 9.6 71.9 3.0 62.8 10.1 AD-953442.1 89.4 4.5 78.2 5.5 99.8 20.9 90.2 7.4 69.0 13.2 95.2 19.1 119.3 10.1 77.5 19.9 AD-953449.1 81.5 5.9 78.3 5.3 95.1 23.3 90.1 7.6 82.6 15.4 72.5 14.2 113.9 14.0 93.2 12.8 AD-953510.1 94.1 6.6 78.6 6.2 85.2 8.5 88.6 4.2 62.7 11.1 74.3 3.6 73.1 12.6 30.5 6.0 AD-953514.1 98.5 10.4 78.8 9.3 95.6 4.4 89.4 4.6 80.1 19.3 89.0 3.5 118.7 18.7 94.8 19.4 AD-953508.1 96.9 7.3 78.9 7.1 96.5 24.7 98.7 8.7 68.5 15.8 76.9 13.9 94.3 15.5 75.2 11.5 AD-953531.1 70.3 2.0 80.7 17.0 82.8 4.0 90.6 4.3 80.9 15.6 62.5 12.3 98.0 12.4 73.4 7.0 AD-953427.1 105.8 12.1 81.1 4.6 91.0 25.6 90.0 3.7 65.3 3.7 100.2 12.6 109.0 17.8 66.6 15.2 AD-953512.1 94.6 2.1 83.9 4.5 91.3 10.6 96.7 7.7 84.8 13.3 84.0 11.0 129.5 10.3 88.3 15.8 AD-953533.1 102.2 9.2 85.2 21.1 82.6 15.3 108.6 13.4 85.5 12.3 66.0 13.3 89.2 14.1 73.0 12.3 AD-953464.1 76.9 3.8 86.1 6.5 104.6 21.8 99.9 10.3 110.5 10.6 74.5 5.0 87.7 18.7 111.9 6.1 AD-953542.1 51.8 2.5 86.3 24.8 66.0 11.3 98.1 17.1 54.8 9.9 69.8 2.3 95.8 13.9 104.4 4.4 AD-953426.1 102.2 10.9 87.8 5.7 103.4 24.0 97.1 2.0 67.3 5.4 120.7 18.0 117.5 16.3 74.5 27.0 AD-953515.1 92.6 5.7 88.4 6.2 84.6 21.4 98.2 9.4 95.5 21.8 84.9 10.3 124.9 28.4 86.9 4.2 AD-953487.1 99.3 9.8 88.7 12.4 90.1 13.7 95.1 3.2 106.9 17.5 88.1 5.6 118.9 35.4 57.1 14.7 AD-953521.1 104.2 20.4 89.0 1.5 109.1 13.9 96.2 9.4 76.6 12.8 89.5 10.4 131.6 23.9 81.8 10.5 AD-953425.1 81.5 13.2 90.2 4.0 99.5 24.2 94.8 6.2 63.6 4.3 114.5 27.3 142.3 60.9 75.3 25.7 AD-953536.1 75.4 13.1 90.2 23.7 86.6 4.9 98.4 8.1 75.0 8.5 58.1 20.2 120.5 32.6 96.6 11.8 AD-953465.1 97.2 10.0 91.4 6.6 114.1 30.7 91.7 5.7 92.3 7.4 78.6 6.8 99.2 29.1 131.0 23.9 AD-953552.1 82.6 7.9 92.5 22.4 88.2 4.8 102.3 6.4 86.4 15.8 85.2 16.6 55.7 16.7 89.0 12.2 AD-953528.1 87.5 15.0 93.5 11.1 83.9 8.8 85.7 4.5 111.2 40.7 69.5 16.7 142.9 10.9 103.3 8.0 AD-953519.1 107.5 2.3 93.5 4.8 96.1 5.2 91.0 3.3 92.7 9.0 113.7 6.9 146.7 24.6 121.8 38.4 AD-953486.1 105.6 3.7 94.2 5.6 103.9 8.6 96.7 6.1 75.6 1.5 106.1 34.1 83.7 17.1 78.1 21.7 AD-953522.1 97.7 9.0 94.3 4.2 100.0 16.6 99.0 8.4 98.8 21.3 87.2 16.4 106.8 15.9 84.1 7.4 AD-953530.1 76.4 4.7 94.3 19.0 90.2 8.9 93.8 9.9 72.4 16.7 67.0 12.1 93.5 11.7 93.3 11.8 AD-953513.1 104.1 3.5 95.5 7.9 92.8 9.4 103.5 10.8 90.2 28.8 76.6 6.8 144.6 24.5 101.7 12.6 AD-953441.1 97.5 8.0 95.5 5.0 114.8 18.2 106.5 8.7 63.8 4.1 105.6 4.8 125.4 24.0 80.4 27.5 AD-953440.1 87.8 6.4 95.5 12.2 112.8 26.2 97.9 1.2 83.3 7.9 106.5 13.4 118.0 10.0 75.4 27.2 AD-953518.1 91.0 4.9 96.1 3.4 99.6 2.8 93.9 12.4 96.6 24.0 91.4 16.4 126.5 19.7 108.8 16.1 AD-953520.1 92.8 7.3 96.1 3.8 99.9 18.5 99.9 7.3 91.8 18.0 89.3 13.6 140.3 21.3 96.0 17.7 AD-953532.1 92.3 7.2 99.5 16.4 96.9 11.9 93.0 4.9 87.9 12.7 67.9 9.0 89.9 24.8 70.6 13.2 AD-953548.1 85.7 8.0 100.5 20.0 89.8 7.6 99.4 7.1 58.8 8.9 67.7 24.6 80.1 6.0 78.0 9.0 AD-953527.1 91.5 2.8 101.9 15.1 92.3 14.5 102.6 4.5 113.8 41.2 69.2 18.2 132.4 36.0 108.0 10.6 AD-953551.1 91.4 4.5 103.0 24.3 94.8 1.1 101.1 5.4 130.4 41.4 78.2 20.9 65.8 13.8 102.2 11.8 AD-953553.1 93.9 1.2 103.2 25.6 93.3 6.6 100.2 4.2 88.5 10.1 93.5 20.0 65.1 15.8 93.0 13.6 AD-953547.1 89.1 3.6 104.0 18.7 94.3 7.6 97.6 3.0 65.0 6.4 60.7 16.1 85.8 9.7 86.6 8.6 AD-953526.1 88.7 5.7 110.0 23.2 95.7 3.2 100.8 12.4 117.9 52.9 75.4 16.8 115.9 12.6 102.1 10.6 AD-953549.1 94.4 8.8 113.8 25.9 70.6 23.4 106.6 12.7 54.1 8.2 55.2 18.7 85.6 31.6 81.4 15.2 AD-953534.1 102.5 8.5 117.9 27.3 95.0 14.1 79.9 5.5 137.0 23.5 81.4 20.8 141.1 19.1 103.4 12.1 AD-953543.1 99.3 3.5 118.0 17.9 89.7 4.0 96.9 4.6 111.5 41.1 99.7 30.4 123.8 17.6 104.4 15.0 AD-953550.1 96.0 5.8 121.6 19.6 98.9 12.3 99.0 9.0 119.6 20.5 101.5 20.0 52.7 10.9 93.6 13.1

The results of the multi-dose screen in African green monkey kidney cells (Cos-7) transfected with Cynomolgus monkey VEGF-A with a set of exemplary rat VEGF-A siRNAs are shown in Table 7A (correspond to siRNAs in Table 5A and Table 5B). The results of the multi-dose screen in Cos-7 cells transfected with mouse VEGF-A with a set of exemplary rat VEGF-A siRNAs are shown in Table 7B (correspond to siRNAs in Table 5A and Table 5B). The multi-dose experiments were performed at 10 nM, 1 nM, and 0.1 nM final duplex concentrations and the data are expressed as percent message remaining relative to non-targeting control.

TABLE 7A Cynomolgus monkey VEGF-A in vitro multi-dose screen with one set of exemplary rat VEGF-A siRNAs Dose Duplex Name Average StDev Dose Unit AD-579911.1 21.79 0.50 10 nM AD-579912.1 20.38 3.81 10 nM AD-579913.1 16.71 8.39 10 nM AD-579914.1 45.98 4.11 10 nM AD-579915.1 35.03 7.59 10 nM AD-579916.1 12.14 1.83 10 nM AD-579917.1 25.40 3.24 10 nM AD-579918.1 26.56 2.53 10 nM AD-579919.1 10.58 2.15 10 nM AD-579921.1 12.64 2.73 10 nM AD-579922.1 13.78 0.77 10 nM AD-579923.1 17.37 1.62 10 nM AD-579924.1 49.49 4.24 10 nM AD-579925.1 10.62 1.31 10 nM AD-579926.1 13.23 2.83 10 nM AD-579927.1 25.34 3.60 10 nM AD-579929.1 26.33 1.74 10 nM AD-579930.1 81.28 12.54 10 nM AD-579931.1 41.52 7.38 10 nM AD-579932.1 12.14 3.92 10 nM AD-579933.1 30.75 3.29 10 nM AD-579934.1 186.16 29.32 10 nM AD-579935.1 67.60 6.57 10 nM AD-579936.1 81.10 20.59 10 nM AD-579937.1 25.95 3.27 10 nM AD-579938.1 60.65 8.90 10 nM AD-579939.1 72.01 14.89 10 nM AD-579940.1 96.52 8.39 10 nM AD-579941.1 106.40 13.04 10 nM AD-579942.1 61.87 12.12 10 nM AD-579943.1 12.46 4.75 10 nM AD-579944.1 28.49 4.13 10 nM AD-579945.1 70.53 8.74 10 nM AD-579946.1 24.47 6.19 10 nM AD-579947.1 68.58 4.38 10 nM AD-579948.1 77.63 5.92 10 nM AD-579949.1 96.51 8.80 10 nM AD-579950.1 230.10 21.51 10 nM AD-579951.1 66.36 16.79 10 nM AD-579953.1 18.42 5.42 10 nM AD-579954.1 21.24 4.31 10 nM AD-579955.1 69.32 6.99 10 nM AD-579956.1 99.49 25.18 10 nM AD-579957.1 49.05 14.80 10 nM AD-579958.1 88.74 8.73 10 nM AD-579959.1 100.41 9.53 10 nM AD-579960.1 39.31 3.68 10 nM AD-579961.1 96.81 13.20 10 nM AD-579962.1 53.43 6.50 10 nM AD-579963.1 21.24 3.25 10 nM AD-579964.1 17.83 7.13 10 nM AD-579965.1 92.27 14.22 10 nM AD-579966.1 88.97 9.79 10 nM AD-579967.1 47.49 7.04 10 nM AD-579968.1 76.96 8.70 10 nM AD-579969.1 90.81 8.30 10 nM AD-579970.1 104.42 26.45 10 nM AD-579971.1 14.37 6.47 10 nM AD-579972.1 57.43 2.61 10 nM AD-579973.1 103.95 3.97 10 nM AD-579974.1 81.04 3.86 10 nM AD-579975.1 33.28 7.01 10 nM AD-579976.1 79.49 2.88 10 nM AD-579977.1 80.67 6.29 10 nM AD-579978.1 66.72 3.59 10 nM AD-579979.1 92.24 3.77 10 nM AD-579980.1 25.30 3.08 10 nM AD-579981.1 48.69 11.03 10 nM AD-579982.1 49.50 15.70 10 nM AD-579983.1 10.78 4.14 10 nM AD-579984.1 16.79 1.90 10 nM AD-579985.1 11.35 0.96 10 nM AD-579986.1 33.51 4.42 10 nM AD-579987.1 26.04 3.89 10 nM AD-579988.1 30.67 3.01 10 nM AD-579989.1 33.51 6.72 10 nM AD-579990.1 72.51 4.50 10 nM AD-579992.1 61.70 6.74 10 nM AD-579993.1 92.69 10.40 10 nM AD-579995.1 30.40 4.51 10 nM AD-579996.1 94.02 11.86 10 nM AD-579997.1 34.79 5.90 10 nM AD-579998.1 31.10 6.52 10 nM AD-579999.1 137.05 8.03 10 nM AD-580000.1 162.84 26.73 10 nM AD-580001.1 125.00 7.31 10 nM AD-580002.1 186.90 14.34 10 nM AD-579911.1 34.63 4.90 1 nM AD-579912.1 31.99 3.89 1 nM AD-579913.1 36.38 2.19 1 nM AD-579914.1 47.66 5.91 1 nM AD-579915.1 33.92 0.98 1 nM AD-579916.1 25.64 5.85 1 nM AD-579917.1 38.41 8.43 1 nM AD-579918.1 37.12 9.78 1 nM AD-579919.1 13.33 2.62 1 nM AD-579921.1 24.01 3.06 1 nM AD-579922.1 21.65 1.29 1 nM AD-579923.1 32.00 1.21 1 nM AD-579924.1 74.46 5.43 1 nM AD-579925.1 27.19 4.59 1 nM AD-579926.1 23.17 2.37 1 nM AD-579927.1 44.62 10.37 1 nM AD-579929.1 32.40 3.90 1 nM AD-579930.1 82.85 10.31 1 nM AD-579931.1 53.35 11.21 1 nM AD-579932.1 25.97 4.61 1 nM AD-579933.1 64.26 11.63 1 nM AD-579934.1 110.08 21.79 1 nM AD-579935.1 75.45 3.94 1 nM AD-579936.1 88.42 7.56 1 nM AD-579937.1 25.63 2.94 1 nM AD-579938.1 67.82 2.00 1 nM AD-579939.1 69.81 7.16 1 nM AD-579940.1 99.17 5.18 1 nM AD-579941.1 108.34 7.84 1 nM AD-579942.1 70.36 12.47 1 nM AD-579943.1 26.50 3.47 1 nM AD-579944.1 35.87 5.32 1 nM AD-579945.1 82.17 15.84 1 nM AD-579946.1 28.27 2.22 1 nM AD-579947.1 77.96 16.94 1 nM AD-579948.1 79.99 3.99 1 nM AD-579949.1 108.10 12.09 1 nM AD-579950.1 134.00 16.20 1 nM AD-579951.1 66.05 9.65 1 nM AD-579953.1 27.50 5.68 1 nM AD-579954.1 20.74 3.51 1 nM AD-579955.1 74.44 17.09 1 nM AD-579956.1 113.45 9.64 1 nM AD-579957.1 73.78 1.80 1 nM AD-579958.1 94.21 11.02 1 nM AD-579959.1 95.73 17.31 1 nM AD-579960.1 37.92 6.36 1 nM AD-579961.1 95.86 5.08 1 nM AD-579962.1 79.16 6.58 1 nM AD-579963.1 22.85 4.33 1 nM AD-579964.1 23.33 2.88 1 nM AD-579965.1 96.34 24.13 1 nM AD-579966.1 85.86 12.83 1 nM AD-579967.1 59.03 6.42 1 nM AD-579968.1 72.33 7.43 1 nM AD-579969.1 86.79 3.22 1 nM AD-579970.1 100.80 8.64 1 nM AD-579971.1 12.62 2.38 1 nM AD-579972.1 52.06 7.34 1 nM AD-579973.1 96.38 18.09 1 nM AD-579974.1 90.54 6.77 1 nM AD-579975.1 54.49 4.18 1 nM AD-579976.1 83.49 18.12 1 nM AD-579977.1 86.21 10.51 1 nM AD-579978.1 73.79 28.62 1 nM AD-579979.1 102.91 17.40 1 nM AD-579980.1 22.61 1.11 1 nM AD-579981.1 62.79 8.71 1 nM AD-579982.1 57.63 7.73 1 nM AD-579983.1 17.63 2.73 1 nM AD-579984.1 25.44 1.54 1 nM AD-579985.1 20.01 6.16 1 nM AD-579986.1 45.92 2.90 1 nM AD-579987.1 35.60 5.94 1 nM AD-579988.1 41.30 3.29 1 nM AD-579989.1 35.25 1.19 1 nM AD-579990.1 75.79 12.51 1 nM AD-579992.1 70.19 10.59 1 nM AD-579993.1 81.86 8.27 1 nM AD-579995.1 32.90 4.47 1 nM AD-579996.1 91.93 9.06 1 nM AD-579997.1 54.91 2.41 1 nM AD-579998.1 35.62 5.31 1 nM AD-579999.1 105.80 11.56 1 nM AD-580000.1 151.28 8.84 1 nM AD-580001.1 117.48 17.60 1 nM AD-580002.1 148.64 32.44 1 nM AD-579911.1 60.59 5.99 0.1 nM AD-579912.1 58.68 4.12 0.1 nM AD-579913.1 64.23 5.02 0.1 nM AD-579914.1 74.48 8.13 0.1 nM AD-579915.1 56.70 3.72 0.1 nM AD-579916.1 41.93 5.43 0.1 nM AD-579917.1 78.90 9.40 0.1 nM AD-579918.1 66.57 10.31 0.1 nM AD-579919.1 29.08 2.79 0.1 nM AD-579921.1 59.28 4.60 0.1 nM AD-579922.1 56.67 8.89 0.1 nM AD-579923.1 75.33 12.81 0.1 nM AD-579924.1 100.79 20.47 0.1 nM AD-579925.1 72.05 13.30 0.1 nM AD-579926.1 63.98 8.75 0.1 nM AD-579927.1 78.12 11.58 0.1 nM AD-579929.1 67.27 19.11 0.1 nM AD-579930.1 96.27 17.88 0.1 nM AD-579931.1 75.81 15.09 0.1 nM AD-579932.1 61.06 2.54 0.1 nM AD-579933.1 85.95 8.05 0.1 nM AD-579934.1 100.61 4.77 0.1 nM AD-579935.1 88.00 10.57 0.1 nM AD-579936.1 88.13 9.64 0.1 nM AD-579937.1 55.87 4.99 0.1 nM AD-579938.1 90.35 12.54 0.1 nM AD-579939.1 100.22 5.50 0.1 nM AD-579940.1 117.60 9.00 0.1 nM AD-579941.1 100.53 13.72 0.1 nM AD-579942.1 100.72 12.00 0.1 nM AD-579943.1 41.23 3.38 0.1 nM AD-579944.1 73.67 9.65 0.1 nM AD-579945.1 101.57 13.70 0.1 nM AD-579946.1 42.00 1.99 0.1 nM AD-579947.1 88.52 29.57 0.1 nM AD-579948.1 94.78 13.34 0.1 nM AD-579949.1 106.50 4.63 0.1 nM AD-579950.1 112.38 10.41 0.1 nM AD-579951.1 81.88 7.61 0.1 nM AD-579953.1 65.43 5.35 0.1 nM AD-579954.1 29.05 3.98 0.1 nM AD-579955.1 101.64 8.13 0.1 nM AD-579956.1 109.12 12.60 0.1 nM AD-579957.1 95.19 6.14 0.1 nM AD-579958.1 99.02 14.55 0.1 nM AD-579959.1 96.08 7.16 0.1 nM AD-579960.1 65.43 20.16 0.1 nM AD-579961.1 89.02 11.37 0.1 nM AD-579962.1 107.63 11.06 0.1 nM AD-579963.1 54.55 6.58 0.1 nM AD-579964.1 37.19 5.60 0.1 nM AD-579965.1 93.32 9.87 0.1 nM AD-579966.1 93.21 13.54 0.1 nM AD-579967.1 78.97 5.07 0.1 nM AD-579968.1 83.13 8.48 0.1 nM AD-579969.1 97.62 21.40 0.1 nM AD-579970.1 102.49 8.36 0.1 nM AD-579971.1 22.84 3.85 0.1 nM AD-579972.1 83.71 13.31 0.1 nM AD-579973.1 97.02 12.16 0.1 nM AD-579974.1 87.05 8.86 0.1 nM AD-579975.1 85.49 9.25 0.1 nM AD-579976.1 89.61 16.73 0.1 nM AD-579977.1 93.52 7.83 0.1 nM AD-579978.1 87.68 5.19 0.1 nM AD-579979.1 93.22 6.02 0.1 nM AD-579980.1 39.04 7.41 0.1 nM AD-579981.1 75.66 11.68 0.1 nM AD-579982.1 90.44 10.96 0.1 nM AD-579983.1 50.03 2.51 0.1 nM AD-579984.1 58.06 3.26 0.1 nM AD-579985.1 33.30 3.60 0.1 nM AD-579986.1 74.91 4.29 0.1 nM AD-579987.1 71.15 8.39 0.1 nM AD-579988.1 81.86 6.80 0.1 nM AD-579989.1 59.63 7.75 0.1 nM AD-579990.1 95.13 9.94 0.1 nM AD-579992.1 94.26 5.07 0.1 nM AD-579993.1 99.78 15.90 0.1 nM AD-579995.1 73.91 9.98 0.1 nM AD-579996.1 101.07 13.27 0.1 nM AD-579997.1 89.33 7.03 0.1 nM AD-579998.1 60.03 5.93 0.1 nM AD-579999.1 109.04 17.76 0.1 nM AD-580000.1 108.28 13.14 0.1 nM AD-580001.1 88.49 11.96 0.1 nM AD-580002.1 122.07 18.09 0.1 nM

TABLE 7B Mouse VEGF-A in vitro multi-dose screen with one set of exemplary rat VEGF-A siRNAs Dose Duplex Name Average StDev Dose Unit AD-579911.1 21.27 3.91 10 nM AD-579912.1 16.53 5.79 10 nM AD-579913.1 16.73 4.22 10 nM AD-579914.1 16.51 1.15 10 nM AD-579915.1 18.33 6.95 10 nM AD-579916.1 25.18 1.82 10 nM AD-579917.1 12.22 1.48 10 nM AD-579918.1 11.07 2.65 10 nM AD-579919.1 12.63 1.67 10 nM AD-579921.1 11.45 2.37 10 nM AD-579922.1 14.90 3.21 10 nM AD-579923.1 13.67 3.59 10 nM AD-579924.1 26.37 2.49 10 nM AD-579925.1 10.16 1.07 10 nM AD-579926.1 14.22 0.82 10 nM AD-579927.1 32.32 5.45 10 nM AD-579929.1 22.15 6.29 10 nM AD-579930.1 74.90 13.83 10 nM AD-579931.1 53.69 19.88 10 nM AD-579932.1 24.54 2.62 10 nM AD-579933.1 20.31 4.52 10 nM AD-579934.1 36.61 3.37 10 nM AD-579935.1 24.55 4.10 10 nM AD-579936.1 22.50 4.65 10 nM AD-579937.1 32.75 3.55 10 nM AD-579938.1 13.39 2.36 10 nM AD-579939.1 4.62 0.19 10 nM AD-579940.1 3.68 1.38 10 nM AD-579941.1 8.87 1.96 10 nM AD-579942.1 3.71 1.54 10 nM AD-579943.1 11.89 1.92 10 nM AD-579944.1 12.26 1.40 10 nM AD-579945.1 16.73 1.86 10 nM AD-579946.1 18.67 5.27 10 nM AD-579947.1 6.39 1.71 10 nM AD-579948.1 27.00 3.21 10 nM AD-579949.1 35.57 2.31 10 nM AD-579950.1 53.80 4.29 10 nM AD-579951.1 17.89 3.43 10 nM AD-579953.1 24.48 3.96 10 nM AD-579954.1 21.78 1.63 10 nM AD-579955.1 30.54 3.58 10 nM AD-579956.1 27.17 1.58 10 nM AD-579957.1 16.61 1.30 10 nM AD-579958.1 53.03 4.43 10 nM AD-579959.1 40.73 3.55 10 nM AD-579960.1 41.68 1.59 10 nM AD-579961.1 29.58 7.59 10 nM AD-579962.1 19.90 1.25 10 nM AD-579963.1 14.55 3.30 10 nM AD-579964.1 23.53 4.62 10 nM AD-579965.1 5.85 1.35 10 nM AD-579966.1 3.37 0.46 10 nM AD-579967.1 8.12 3.03 10 nM AD-579968.1 15.39 2.69 10 nM AD-579969.1 41.77 5.27 10 nM AD-579970.1 35.40 4.42 10 nM AD-579971.1 9.46 1.20 10 nM AD-579972.1 13.66 4.21 10 nM AD-579973.1 15.20 4.13 10 nM AD-579974.1 3.60 1.49 10 nM AD-579975.1 3.08 0.33 10 nM AD-579976.1 7.90 0.37 10 nM AD-579977.1 22.23 1.40 10 nM AD-579978.1 15.03 2.22 10 nM AD-579979.1 29.83 5.08 10 nM AD-579980.1 25.34 2.41 10 nM AD-579981.1 20.80 1.40 10 nM AD-579982.1 20.12 8.04 10 nM AD-579983.1 22.13 4.26 10 nM AD-579984.1 11.34 1.30 10 nM AD-579985.1 10.84 0.91 10 nM AD-579986.1 22.36 1.53 10 nM AD-579987.1 26.77 5.29 10 nM AD-579988.1 25.91 6.97 10 nM AD-579989.1 17.89 1.51 10 nM AD-579990.1 29.33 3.43 10 nM AD-579992.1 19.38 7.67 10 nM AD-579993.1 23.19 0.70 10 nM AD-579995.1 4.97 1.31 10 nM AD-579996.1 63.08 6.91 10 nM AD-579997.1 26.46 4.46 10 nM AD-579998.1 17.58 4.81 10 nM AD-579999.1 33.79 5.04 10 nM AD-580000.1 28.19 4.07 10 nM AD-580001.1 23.17 4.55 10 nM AD-580002.1 51.58 4.73 10 nM AD-579911.1 29.66 4.64 1 nM AD-579912.1 26.43 3.94 1 nM AD-579913.1 40.12 5.03 1 nM AD-579914.1 24.93 2.78 1 nM AD-579915.1 34.06 4.64 1 nM AD-579916.1 45.46 4.47 1 nM AD-579917.1 19.16 2.98 1 nM AD-579918.1 16.77 1.65 1 nM AD-579919.1 17.09 1.57 1 nM AD-579921.1 21.90 0.76 1 nM AD-579922.1 24.16 3.67 1 nM AD-579923.1 28.64 5.98 1 nM AD-579924.1 54.14 8.12 1 nM AD-579925.1 27.88 2.54 1 nM AD-579926.1 29.26 2.01 1 nM AD-579927.1 44.60 5.25 1 nM AD-579929.1 31.53 4.15 1 nM AD-579930.1 93.72 13.68 1 nM AD-579931.1 54.78 5.31 1 nM AD-579932.1 46.12 4.65 1 nM AD-579933.1 27.01 5.56 1 nM AD-579934.1 40.35 6.96 1 nM AD-579935.1 31.13 5.41 1 nM AD-579936.1 29.73 3.71 1 nM AD-579937.1 44.09 4.36 1 nM AD-579938.1 21.66 3.66 1 nM AD-579939.1 4.58 0.75 1 nM AD-579940.1 3.56 0.91 1 nM AD-579941.1 7.55 2.31 1 nM AD-579942.1 5.56 2.57 1 nM AD-579943.1 19.30 3.06 1 nM AD-579944.1 18.70 1.80 1 nM AD-579945.1 26.86 1.35 1 nM AD-579946.1 21.86 3.32 1 nM AD-579947.1 9.84 0.95 1 nM AD-579948.1 26.84 1.26 1 nM AD-579949.1 57.75 9.08 1 nM AD-579950.1 54.60 4.99 1 nM AD-579951.1 23.18 1.60 1 nM AD-579953.1 35.85 4.89 1 nM AD-579954.1 33.37 3.57 1 nM AD-579955.1 43.72 4.02 1 nM AD-579956.1 40.52 0.43 1 nM AD-579957.1 22.05 1.37 1 nM AD-579958.1 63.71 6.37 1 nM AD-579959.1 54.17 7.09 1 nM AD-579960.1 45.58 4.07 1 nM AD-579961.1 40.91 2.43 1 nM AD-579962.1 28.65 3.39 1 nM AD-579963.1 22.65 6.61 1 nM AD-579964.1 28.01 5.38 1 nM AD-579965.1 3.88 1.04 1 nM AD-579966.1 4.08 1.00 1 nM AD-579967.1 14.72 0.97 1 nM AD-579968.1 19.71 4.24 1 nM AD-579969.1 54.35 7.63 1 nM AD-579970.1 52.04 1.90 1 nM AD-579971.1 13.28 3.80 1 nM AD-579972.1 22.49 5.22 1 nM AD-579973.1 23.49 2.63 1 nM AD-579974.1 6.50 0.84 1 nM AD-579975.1 3.65 1.14 1 nM AD-579976.1 10.00 2.25 1 nM AD-579977.1 30.06 5.34 1 nM AD-579978.1 21.05 2.77 1 nM AD-579979.1 54.05 6.94 1 nM AD-579980.1 26.38 3.38 1 nM AD-579981.1 22.68 2.02 1 nM AD-579982.1 39.84 3.32 1 nM AD-579983.1 34.64 6.02 1 nM AD-579984.1 16.95 2.53 1 nM AD-579985.1 31.39 1.48 1 nM AD-579986.1 27.74 3.81 1 nM AD-579987.1 51.76 4.91 1 nM AD-579988.1 29.45 2.77 1 nM AD-579989.1 21.33 2.08 1 nM AD-579990.1 47.89 1.76 1 nM AD-579992.1 30.71 4.73 1 nM AD-579993.1 35.91 7.25 1 nM AD-579995.1 6.70 0.49 1 nM AD-579996.1 74.23 4.02 1 nM AD-579997.1 51.99 10.00 1 nM AD-579998.1 20.92 4.64 1 nM AD-579999.1 40.31 1.99 1 nM AD-580000.1 38.17 1.63 1 nM AD-580001.1 28.86 5.37 1 nM AD-580002.1 51.52 7.91 1 nM AD-579911.1 59.37 8.87 0.1 nM AD-579912.1 59.39 5.09 0.1 nM AD-579913.1 64.08 23.73 0.1 nM AD-579914.1 49.48 3.64 0.1 nM AD-579915.1 56.56 6.31 0.1 nM AD-579916.1 96.79 16.03 0.1 nM AD-579917.1 45.36 7.75 0.1 nM AD-579918.1 57.38 4.03 0.1 nM AD-579919.1 55.62 5.60 0.1 nM AD-579921.1 53.70 9.76 0.1 nM AD-579922.1 62.44 4.87 0.1 nM AD-579923.1 63.54 9.06 0.1 nM AD-579924.1 92.45 9.57 0.1 nM AD-579925.1 61.94 18.45 0.1 nM AD-579926.1 70.86 8.69 0.1 nM AD-579927.1 78.15 23.69 0.1 nM AD-579929.1 71.46 14.26 0.1 nM AD-579930.1 90.78 6.27 0.1 nM AD-579931.1 68.75 9.57 0.1 nM AD-579932.1 77.69 9.74 0.1 nM AD-579933.1 51.36 9.29 0.1 nM AD-579934.1 77.13 7.91 0.1 nM AD-579935.1 49.67 7.46 0.1 nM AD-579936.1 44.13 6.15 0.1 nM AD-579937.1 77.96 4.22 0.1 nM AD-579938.1 29.19 7.09 0.1 nM AD-579939.1 10.14 1.04 0.1 nM AD-579940.1 6.98 3.06 0.1 nM AD-579941.1 7.68 1.31 0.1 nM AD-579942.1 8.97 1.33 0.1 nM AD-579943.1 37.14 5.93 0.1 nM AD-579944.1 39.47 3.54 0.1 nM AD-579945.1 54.76 10.45 0.1 nM AD-579946.1 54.38 7.47 0.1 nM AD-579947.1 22.79 5.03 0.1 nM AD-579948.1 38.00 6.15 0.1 nM AD-579949.1 80.25 7.57 0.1 nM AD-579950.1 73.79 5.42 0.1 nM AD-579951.1 47.34 5.57 0.1 nM AD-579953.1 75.21 13.06 0.1 nM AD-579954.1 49.75 4.87 0.1 nM AD-579955.1 84.04 10.95 0.1 nM AD-579956.1 73.48 18.05 0.1 nM AD-579957.1 37.29 3.15 0.1 nM AD-579958.1 96.73 8.67 0.1 nM AD-579959.1 86.90 10.17 0.1 nM AD-579960.1 71.64 12.07 0.1 nM AD-579961.1 86.22 11.71 0.1 nM AD-579962.1 59.52 4.17 0.1 nM AD-579963.1 53.65 1.36 0.1 nM AD-579964.1 60.89 5.53 0.1 nM AD-579965.1 10.37 1.33 0.1 nM AD-579966.1 9.37 1.18 0.1 nM AD-579967.1 33.20 1.60 0.1 nM AD-579968.1 36.38 8.49 0.1 nM AD-579969.1 83.90 5.81 0.1 nM AD-579970.1 104.17 11.38 0.1 nM AD-579971.1 29.04 4.62 0.1 nM AD-579972.1 47.77 5.87 0.1 nM AD-579973.1 67.38 5.17 0.1 nM AD-579974.1 21.80 3.23 0.1 nM AD-579975.1 10.78 2.10 0.1 nM AD-579976.1 20.60 3.17 0.1 nM AD-579977.1 86.60 5.20 0.1 nM AD-579978.1 53.36 3.32 0.1 nM AD-579979.1 92.09 11.37 0.1 nM AD-579980.1 52.60 2.36 0.1 nM AD-579981.1 46.23 7.60 0.1 nM AD-579982.1 94.15 11.36 0.1 nM AD-579983.1 76.94 11.93 0.1 nM AD-579984.1 34.98 2.84 0.1 nM AD-579985.1 69.50 9.11 0.1 nM AD-579986.1 62.04 6.78 0.1 nM AD-579987.1 89.22 9.48 0.1 nM AD-579988.1 72.80 14.40 0.1 nM AD-579989.1 52.85 3.85 0.1 nM AD-579990.1 94.01 8.85 0.1 nM AD-579992.1 67.82 6.38 0.1 nM AD-579993.1 81.30 15.42 0.1 nM AD-579995.1 19.72 5.72 0.1 nM AD-579996.1 97.03 10.38 0.1 nM AD-579997.1 87.06 12.93 0.1 nM AD-579998.1 52.54 4.98 0.1 nM AD-579999.1 71.80 3.62 0.1 nM AD-580000.1 75.04 9.15 0.1 nM AD-580001.1 59.52 8.32 0.1 nM AD-580002.1 90.07 10.26 0.1 nM

The results of the multi-dose screen in primary human hepatocytes transfected with one set of exemplary human VEGF-A siRNAs is shown in Table 9A (correspond to siRNAs in Table 8A and 8B) The multi-dose experiments were performed at 50 nM, 10 nM, 1 nM, and 0.1 nM final duplex concentrations and the data are expressed as percent message remaining relative to non-targeting control.

Of the exemplary siRNA duplexes evaluated in Table 9A, 1 achieved a knockdown of VEGF-A of ≥80%, 119 achieved a knockdown of VEGF-A of ≥60%, and 363 achieved a knockdown of VEGF-A of ≥30% when administered at the 50 nM concentration.

Of the exemplary siRNA duplexes evaluated in Table 9A, 2 achieved a knockdown of VEGF-A of ≥80%, 103 achieved a knockdown of VEGF-A of ≥60%, and 364 achieved a knockdown of VEGF-A of ≥30% when administered at the 10 nM concentration.

Of the exemplary siRNA duplexes evaluated in Table 9A, 13 achieved a knockdown of VEGF-A of ≥70%, 52 achieved a knockdown of VEGF-A of ≥60%, and 312 achieved a knockdown of VEGF-A of ≥30% when administered at the 1 nM concentration.

Of the exemplary siRNA duplexes evaluated in Table 9A, 8 achieved a knockdown of VEGF-A of ≥50%, 75 achieved a knockdown of VEGF-A of ≥40%, and 170 achieved a knockdown of VEGF-A of ≥30% when administered at the 0.1 nM concentration.

TABLE 9A VEGF-A endogenous in vitro multi-dose screen following cellular transfection with one set of exemplary human VEGF-A siRNAs DuplexID 50 nM StDev 10 nM StDev 1 nM StDev 0.1 nM StDev AD-1222866.1 70 22 58 9 69 9 60 6 AD-1222867.1 64 14 38 6 55 3 54 2 AD-1222868.1 60 7 42 6 56 8 55 3 AD-1222869.1 61 3 51 1 56 3 56 0 AD-1222870.1 43 7 39 6 47 7 52 4 AD-1222871.1 41 3 38 7 42 2 56 3 AD-1222872.1 54 4 46 9 50 1 56 2 AD-1222873.1 44 1 44 4 46 2 58 1 AD-1222874.1 78 22 66 18 69 4 70 2 AD-1222875.1 56 7 51 5 64 3 59 4 AD-1222876.1 56 9 64 6 71 8 67 4 AD-1222877.1 60 19 50 5 59 7 67 3 AD-1222878.1 64 7 60 2 63 3 72 4 AD-1222879.1 64 3 57 9 52 4 64 9 AD-1222880.1 54 4 53 9 49 4 57 4 AD-1222881.1 58 10 51 2 46 1 63 9 AD-1222882.1 93 12 74 14 93 12 91 12 AD-1222883.1 72 12 59 10 64 7 78 9 AD-1222884.1 63 5 58 8 61 0 79 10 AD-1222885.1 70 12 66 9 69 11 81 4 AD-1222886.1 77 7 76 7 69 3 80 6 AD-1222887.1 62 5 58 11 57 4 70 9 AD-1222888.1 69 6 68 8 64 7 65 4 AD-1222889.1 95 16 81 13 98 11 128 54 AD-1222890.1 88 4 85 11 81 10 97 15 AD-1222891.1 91 15 70 7 77 7 84 16 AD-1222892.1 77 3 75 16 82 22 84 5 AD-1222893.1 84 4 77 14 74 4 85 3 AD-1222894.1 66 6 53 0 61 8 77 10 AD-1222895.1 66 8 68 11 69 6 71 8 AD-1222896.1 99 18 82 25 88 13 124 18 AD-1222897.1 94 10 95 6 76 7 88 14 AD-1222898.1 107 13 93 3 85 10 91 15 AD-1222899.1 98 15 108 5 78 3 90 9 AD-1222900.1 83 14 83 2 82 1 91 8 AD-1222901.1 85 11 81 10 90 15 82 11 AD-1222902.1 71 11 70 6 78 7 87 11 AD-1222903.1 85 5 66 10 80 11 81 6 AD-1222904.1 99 13 87 18 87 9 109 20 AD-1222905.1 109 10 97 14 91 10 103 20 AD-1222906.1 117 6 94 14 85 8 78 3 AD-1222907.1 91 15 85 11 73 20 81 13 AD-1222908.1 82 5 77 11 81 19 90 11 AD-1222909.1 83 8 85 16 62 13 89 8 AD-1222910.1 71 7 78 9 70 2 86 9 AD-1222911.1 129 1 93 17 102 3 116 24 AD-1222912.1 117 11 115 10 98 10 114 7 AD-1222913.1 103 9 89 6 91 5 110 19 AD-1222914.1 72 3 63 3 80 16 91 11 AD-1222915.1 59 11 60 6 80 7 100 8 AD-1222916.1 49 6 42 5 43 12 94 10 AD-1222917.1 61 2 49 6 62 7 85 4 AD-1222918.1 70 12 69 4 77 4 94 4 AD-1222920.1 87 15 71 6 59 5 90 10 AD-1222921.1 104 8 105 6 89 10 103 13 AD-1222922.1 68 7 78 3 88 18 78 14 AD-1222923.1 58 4 64 8 64 5 90 2 AD-1222924.1 82 10 84 13 83 17 100 2 AD-1222925.1 71 7 83 11 84 13 91 0 AD-1222926.1 90 9 87 9 80 2 101 2 AD-1222927.1 95 14 84 3 76 4 103 21 AD-1222928.1 62 4 54 5 54 4 84 3 AD-1222929.1 79 9 61 6 69 2 87 23 AD-1222930.1 59 1 62 1 68 2 95 8 AD-1222931.1 55 4 58 2 64 3 89 16 AD-1222932.1 75 18 87 8 93 11 88 7 AD-1222933.1 40 12 47 5 45 3 87 17 AD-1222934.1 56 8 42 5 48 10 68 8 AD-1222935.1 41 0 41 2 46 4 66 8 AD-1222936.1 39 3 50 2 54 1 73 8 AD-1222937.1 50 13 52 10 63 7 89 9 AD-1222938.1 46 5 67 8 72 4 96 7 AD-1222939.1 37 2 41 7 57 13 82 4 AD-1222940.1 36 6 45 8 47 8 74 17 AD-1222941.1 89 12 76 9 85 6 85 17 AD-1222942.1 44 7 39 3 43 6 67 4 AD-1222943.1 81 17 77 6 85 5 100 5 AD-1222944.1 53 11 50 4 59 14 85 9 AD-1222945.1 74 12 73 0 84 23 92 4 AD-1222946.1 37 3 44 4 76 7 89 7 AD-1222947.1 31 4 31 3 52 3 54 4 AD-1222948.1 58 12 60 6 88 14 111 52 AD-1222949.1 52 12 52 11 63 17 109 13 AD-1222950.1 52 18 51 1 72 7 113 21 AD-1222951.1 37 12 34 5 42 1 95 19 AD-1222952.1 78 4 89 6 103 1 137 44 AD-1222953.1 27 6 30 2 57 6 119 47 AD-1222954.1 33 4 40 4 68 12 102 35 AD-1222955.1 38 9 39 3 53 5 116 54 AD-1222956.1 34 6 42 7 58 20 69 5 AD-1222957.1 23 3 30 2 55 20 62 9 AD-1222958.1 21 3 35 15 35 8 59 4 AD-1222959.1 21 2 30 8 39 7 65 1 AD-1222960.1 25 2 32 6 39 1 68 11 AD-1222961.1 32 5 25 1 41 7 54 2 AD-1222962.1 33 3 34 6 34 8 55 5 AD-1222963.1 26 3 24 0 34 13 61 3 AD-1222964.1 37 1 47 7 78 10 87 14 AD-1222965.1 49 0 58 19 81 11 88 3 AD-1222966.1 48 7 51 4 59 6 82 3 AD-1222967.1 36 2 34 2 49 6 67 8 AD-1222968.1 39 3 47 5 53 10 73 7 AD-1222969.1 30 1 28 3 41 11 55 5 AD-1222970.1 18 1 19 2 25 10 41 2 AD-1222971.1 40 5 35 2 44 4 58 4 AD-1222972.1 41 4 47 8 87 7 87 11 AD-1222973.1 28 2 31 10 52 7 71 8 AD-1222974.1 38 4 29 2 57 8 67 11 AD-1222975.1 35 3 28 2 48 15 72 6 AD-1222976.1 24 1 23 5 33 2 49 2 AD-1222977.1 22 1 20 2 28 5 47 4 AD-1222978.1 27 6 24 6 31 7 50 3 AD-1222979.1 52 11 52 0 71 13 103 26 AD-1222980.1 46 5 41 5 65 9 91 2 AD-1222981.1 49 4 41 3 59 11 76 2 AD-1222982.1 35 5 29 1 44 7 64 4 AD-1222983.1 32 4 28 2 35 10 54 1 AD-1222984.1 29 2 25 1 32 1 53 3 AD-1222985.1 37 3 34 7 43 6 57 3 AD-1222986.1 34 2 34 1 65 13 94 23 AD-1222987.1 38 5 35 7 48 10 73 9 AD-1222988.1 31 0 27 1 38 2 59 7 AD-1222989.1 35 2 29 5 42 2 59 4 AD-1222990.1 30 4 34 10 37 7 59 6 AD-1222991.1 26 1 21 2 37 9 59 9 AD-1222992.1 39 5 38 0 49 14 65 1 AD-1222993.1 39 1 36 3 43 5 80 17 AD-1222994.1 46 3 48 6 54 1 82 3 AD-1222995.1 47 5 36 5 67 16 76 7 AD-1222996.1 58 7 50 4 60 4 79 2 AD-1222997.1 40 8 37 5 50 4 53 9 AD-1222998.1 43 8 39 4 51 3 51 7 AD-1222999.1 40 1 43 12 45 2 60 5 AD-1223000.1 55 12 45 4 53 2 69 4 AD-1223001.1 56 7 62 9 77 15 103 15 AD-1223002.1 69 1 65 1 78 6 86 9 AD-1223003.1 52 10 46 10 60 9 72 5 AD-1223004.1 34 2 32 3 41 9 58 4 AD-1223005.1 47 1 51 12 51 12 67 7 AD-1223006.1 35 2 32 2 39 1 61 7 AD-1223007.1 55 7 42 3 54 10 61 6 AD-1223008.1 32 4 29 1 37 1 68 10 AD-1223009.1 40 3 47 7 54 6 97 15 AD-1223010.1 52 2 46 3 58 8 85 6 AD-1223011.1 44 2 39 5 39 7 58 7 AD-1223012.1 32 3 37 4 39 5 61 7 AD-1223013.1 78 9 65 10 60 4 79 4 AD-1223014.1 64 10 50 4 46 7 76 4 AD-1223015.1 38 5 29 3 43 8 64 4 AD-1223016.1 47 11 31 3 35 6 81 17 AD-1223017.1 55 4 52 2 53 8 89 23 AD-1223018.1 39 0 45 12 47 1 75 16 AD-1223019.1 39 5 36 4 46 14 67 2 AD-1223020.1 48 9 47 15 44 9 68 2 AD-1223021.1 50 7 42 5 41 1 68 13 AD-1223022.1 34 4 31 2 37 5 61 13 AD-1223023.1 66 7 56 2 59 9 80 13 AD-1223024.1 55 10 52 6 78 9 115 31 AD-1223025.1 49 4 47 4 60 1 109 9 AD-1223026.1 55 6 55 6 59 14 86 10 AD-1223027.1 66 3 63 10 70 19 90 1 AD-1223028.1 54 6 48 6 53 3 82 15 AD-1223029.1 52 3 50 8 62 1 81 7 AD-1223030.1 53 7 49 7 48 2 74 12 AD-1223031.1 44 8 38 3 60 5 88 14 AD-1223032.1 51 2 53 2 57 4 88 8 AD-1223033.1 46 2 49 15 63 10 77 18 AD-1223034.1 53 18 36 4 57 19 81 11 AD-1223035.1 50 2 49 15 50 9 65 4 AD-1223036.1 40 3 34 6 43 5 77 31 AD-1223037.1 42 3 33 5 37 6 55 8 AD-1223038.1 30 4 32 1 29 1 62 2 AD-1223039.1 67 3 48 8 68 12 74 14 AD-1223040.1 61 1 54 13 56 9 73 10 AD-1223041.1 57 7 44 4 65 17 91 7 AD-1223042.1 62 32 46 6 55 13 71 4 AD-1223043.1 41 5 40 3 46 8 79 6 AD-1223044.1 53 17 39 2 43 6 73 9 AD-1223045.1 54 0 40 2 44 3 67 9 AD-1223046.1 49 13 43 3 32 3 103 1 AD-1223047.1 55 4 41 4 26 7 97 8 AD-1223048.1 43 2 39 3 25 8 99 14 AD-1223049.1 31 2 35 1 36 10 107 9 AD-1223050.1 32 4 37 3 63 5 106 21 AD-1223051.1 55 4 48 3 74 30 114 16 AD-1223052.1 50 12 39 3 62 4 107 14 AD-1223053.1 37 2 44 6 41 13 90 12 AD-1223054.1 51 23 48 1 28 5 98 11 AD-1223055.1 63 41 44 1 22 2 79 8 AD-1223056.1 49 1 45 1 25 5 83 11 AD-1223057.1 41 13 42 6 39 15 79 11 AD-1223058.1 26 3 33 2 53 25 77 10 AD-1223059.1 32 2 37 0 50 11 71 10 AD-1223060.1 41 13 38 3 50 2 78 2 AD-1223061.1 40 16 43 2 52 5 80 20 AD-1223062.1 52 15 57 4 29 7 99 2 AD-1223063.1 40 8 52 7 31 1 91 10 AD-1223064.1 61 13 60 12 60 14 94 5 AD-1223065.1 51 11 42 5 69 19 72 11 AD-1223066.1 30 7 34 2 57 16 73 16 AD-1223067.1 33 0 49 3 83 21 85 14 AD-1223068.1 44 14 49 7 50 3 71 6 AD-1223069.1 48 3 69 1 49 3 111 5 AD-1223070.1 40 16 66 5 44 18 89 7 AD-1223071.1 38 6 48 12 45 13 89 11 AD-1223072.1 55 8 58 4 82 10 78 9 AD-1223073.1 39 5 47 7 96 9 69 5 AD-1223074.1 38 3 43 2 60 10 68 6 AD-1223075.1 35 2 46 2 55 14 66 8 AD-1223076.1 42 9 67 2 30 2 108 21 AD-1223077.1 43 4 75 5 58 20 99 7 AD-1223078.1 63 9 74 10 79 22 91 6 AD-1223079.1 76 13 52 2 75 10 92 23 AD-1223080.1 39 3 45 4 70 8 69 11 AD-1223081.1 53 14 57 4 76 4 74 14 AD-1223082.1 34 3 43 3 46 7 60 4 AD-1223083.1 41 11 50 0 47 12 53 4 AD-1223084.1 53 11 58 7 44 9 64 4 AD-1223085.1 46 0 60 1 58 7 91 12 AD-1223086.1 57 11 51 1 73 6 67 9 AD-1223087.1 68 3 71 1 87 5 84 17 AD-1223088.1 64 17 61 12 86 1 88 9 AD-1223089.1 35 5 37 4 50 8 71 7 AD-1223090.1 48 11 51 4 56 7 65 2 AD-1223091.1 45 13 54 5 60 15 63 4 AD-1223092.1 48 18 63 5 83 13 86 7 AD-1223093.1 52 5 48 1 79 18 73 1 AD-1223094.1 40 14 52 10 81 13 76 10 AD-1223095.1 41 11 42 1 66 14 65 7 AD-1223096.1 33 5 51 2 81 23 71 4 AD-1223097.1 31 3 48 6 52 2 77 10 AD-1223098.1 31 8 39 3 41 6 57 1 AD-1223099.1 42 15 58 5 56 8 60 1 AD-1223100.1 35 9 55 7 54 5 62 5 AD-1223101.1 52 3 57 8 111 6 62 8 AD-1223102.1 43 13 62 1 75 13 62 6 AD-1223103.1 54 13 50 8 79 7 72 6 AD-1223104.1 36 17 50 12 50 4 53 5 AD-1223105.1 29 4 46 3 50 12 56 2 AD-1223106.1 43 14 50 9 63 22 54 5 AD-1223107.1 31 3 56 2 63 33 68 3 AD-1223108.1 41 13 53 4 55 11 68 5 AD-1223109.1 58 11 69 12 71 2 73 3 AD-1223110.1 65 10 80 11 87 6 83 7 AD-1223111.1 54 14 58 10 58 15 68 4 AD-1223112.1 52 4 82 2 75 14 78 4 AD-1223113.1 55 11 73 6 81 21 84 1 AD-1223114.1 60 8 66 1 83 16 82 1 AD-1223115.1 81 10 73 6 77 1 84 10 AD-1223116.1 79 3 87 8 79 13 82 6 AD-1223117.1 58 6 92 2 74 18 74 5 AD-1223118.1 38 1 82 2 57 2 71 8 AD-1223119.1 51 9 67 5 79 47 78 8 AD-1223120.1 43 1 72 2 53 10 73 3 AD-1223121.1 46 5 68 5 69 5 67 6 AD-1223122.1 61 26 63 8 75 11 78 7 AD-1223123.1 43 12 59 3 59 8 69 4 AD-1223124.1 66 17 76 3 75 14 67 3 AD-1223125.1 54 15 77 3 68 3 64 4 AD-1223126.1 56 10 70 2 53 2 75 16 AD-1223127.1 47 13 70 4 41 7 62 6 AD-1223128.1 52 3 87 1 50 5 75 1 AD-1223129.1 39 1 65 9 55 11 70 5 AD-1223130.1 43 8 66 4 62 8 71 8 AD-1223131.1 48 14 63 5 55 2 70 3 AD-1223132.1 51 8 74 6 75 13 72 2 AD-1223133.1 37 2 61 0 60 4 70 6 AD-1223134.1 41 3 69 2 55 1 71 9 AD-1223135.1 59 20 85 10 54 8 73 4 AD-1223136.1 79 12 134 13 62 9 118 26 AD-1223137.1 65 7 99 30 69 10 127 19 AD-1223138.1 77 19 100 23 68 6 112 28 AD-1223139.1 53 1 127 15 60 15 106 20 AD-1223140.1 59 22 67 7 46 8 98 23 AD-1223141.1 50 6 69 4 52 19 73 22 AD-1223142.1 49 3 63 5 42 11 72 6 AD-1223143.1 45 10 56 11 52 13 68 14 AD-1223144.1 50 8 54 11 60 2 115 5 AD-1223145.1 49 4 74 13 62 19 90 2 AD-1223146.1 81 3 97 35 111 9 107 21 AD-1223147.1 65 15 71 18 62 9 106 13 AD-1223148.1 63 8 74 12 63 11 100 31 AD-1223149.1 49 6 59 13 51 10 100 26 AD-1223150.1 49 2 67 14 47 16 89 21 AD-1223151.1 51 1 62 11 42 5 98 6 AD-1223152.1 67 9 90 20 63 11 123 11 AD-1223153.1 77 3 88 18 109 9 126 12 AD-1223154.1 53 9 46 2 70 8 87 3 AD-1223155.1 55 15 40 4 53 15 129 41 AD-1223156.1 54 9 48 3 48 12 91 13 AD-1223157.1 49 8 53 14 52 11 92 13 AD-1223158.1 45 3 49 9 40 9 84 9 AD-1223159.1 42 7 77 29 59 13 109 7 AD-1223160.1 55 8 55 4 98 7 122 18 AD-1223161.1 49 10 57 9 66 18 118 16 AD-1223162.1 73 5 59 6 52 2 91 13 AD-1223163.1 57 16 59 7 61 13 94 20 AD-1223164.1 58 18 70 8 54 3 120 9 AD-1223165.1 49 13 65 2 50 6 98 6 AD-1223166.1 70 13 72 14 92 9 125 21 AD-1223167.1 78 8 71 9 101 34 119 26 AD-1223168.1 75 17 77 10 98 18 115 15 AD-1223169.1 87 3 57 2 76 27 133 23 AD-1223170.1 87 11 73 12 93 19 85 8 AD-1223171.1 67 20 72 31 70 4 96 12 AD-1223172.1 41 10 44 4 54 9 93 5 AD-1223173.1 62 14 81 21 58 24 116 16 AD-1223174.1 60 14 65 2 96 2 101 9 AD-1223175.1 80 29 61 6 91 19 98 15 AD-1223176.1 77 4 85 6 123 19 109 22 AD-1223177.1 72 14 51 6 70 17 92 1 AD-1223178.1 80 3 77 8 72 16 96 11 AD-1223179.1 82 14 111 9 66 9 81 15 AD-1223180.1 47 9 45 2 54 15 81 12 AD-1223181.1 82 8 76 11 74 17 131 27 AD-1223182.1 58 11 56 13 72 15 80 7 AD-1223183.1 79 20 55 4 94 11 90 9 AD-1223184.1 39 3 38 1 51 4 72 5 AD-1223185.1 63 4 52 10 48 5 76 8 AD-1223186.1 56 17 54 11 48 11 70 4 AD-1223187.1 37 2 39 3 45 14 74 9 AD-1223188.1 32 6 42 7 34 2 68 3 AD-1223189.1 43 14 41 5 56 1 65 12 AD-1223190.1 38 7 41 3 53 14 53 1 AD-1223191.1 55 13 41 7 50 2 56 12 AD-1223192.1 33 3 38 5 43 6 57 6 AD-1223193.1 34 2 30 2 33 9 44 9 AD-1223194.1 45 11 33 6 25 5 48 3 AD-1223195.1 50 19 31 4 39 13 51 4 AD-1223196.1 42 2 29 6 29 11 62 4 AD-1223197.1 42 2 29 0 26 1 59 12 AD-1223198.1 54 10 41 10 61 2 79 8 AD-1223199.1 39 4 36 6 52 16 70 4 AD-1223200.1 35 11 31 2 32 12 57 3 AD-1223201.1 46 18 36 7 32 1 55 7 AD-1223202.1 38 8 31 3 39 3 66 6 AD-1223203.1 39 6 39 6 34 9 66 1 AD-1223204.1 45 4 40 7 45 5 78 2 AD-1223205.1 60 14 54 15 58 4 72 10 AD-1223206.1 43 11 31 5 40 3 57 5 AD-1223207.1 73 2 65 10 57 10 77 12 AD-1223208.1 73 22 68 8 57 10 73 8 AD-1223209.1 48 9 44 10 45 5 73 15 AD-1223210.1 46 9 34 2 43 16 84 9 AD-1223211.1 26 4 40 12 39 7 63 3 AD-1223212.1 46 10 33 3 49 11 68 1 AD-1223213.1 58 6 36 4 46 3 57 2 AD-1223214.1 34 1 35 1 43 6 62 8 AD-1223215.1 41 9 33 6 39 8 61 9 AD-1223216.1 43 8 31 2 47 13 59 4 AD-1223217.1 41 6 39 2 38 7 66 7 AD-1223218.1 31 6 36 4 43 10 65 7 AD-1223219.1 35 7 47 7 48 4 68 1 AD-1223220.1 36 9 39 3 40 11 62 3 AD-1223221.1 43 13 31 0 39 13 58 6 AD-1223222.1 65 27 50 7 57 10 68 9 AD-1223223.1 45 2 39 5 47 2 66 11 AD-1223224.1 32 4 33 5 40 6 60 2 AD-1223225.1 35 8 37 3 31 5 70 9 AD-1223226.1 93 9 62 10 55 5 74 16 AD-1223227.1 100 15 77 12 69 6 69 5 AD-1223228.1 83 5 44 7 44 5 55 3 AD-1223229.1 101 29 59 9 56 7 62 11 AD-1223230.1 85 16 45 6 48 2 52 8 AD-1223231.1 97 18 56 2 57 3 68 12 AD-1223232.1 110 15 79 9 60 2 54 1 AD-1223233.1 95 9 62 4 60 9 58 3 AD-1223234.1 105 8 68 14 72 1 78 13 AD-1223235.1 81 5 64 14 69 12 67 3 AD-1223236.1 83 12 53 6 63 5 90 15 AD-1223237.1 146 33 95 26 82 15 92 16 AD-1223238.1 72 5 68 17 60 5 69 7 AD-1223239.1 75 17 49 3 51 5 67 5 AD-1223240.1 63 2 59 10 53 2 55 9 AD-1223241.1 78 18 55 10 49 5 53 7 AD-1223242.1 77 10 57 6 64 8 70 6 AD-1223243.1 108 1 60 10 70 2 66 11 AD-1223244.1 50 9 62 9 56 9 75 15 AD-1223245.1 59 0 63 12 72 1 79 5 AD-1223246.1 52 4 51 9 44 4 71 9 AD-1223247.1 68 9 58 4 49 3 61 9 AD-1223248.1 80 9 58 8 50 5 54 7 AD-1223249.1 81 16 69 4 96 25 63 13 AD-1223250.1 77 16 72 11 123 20 65 14 AD-1223251.1 58 15 65 7 88 7 60 9 AD-1223252.1 69 12 60 8 80 12 80 21 AD-1223253.1 68 31 48 5 67 13 53 1 AD-1223254.1 49 7 56 12 84 0 54 7 AD-1223255.1 46 5 50 8 59 6 46 8 AD-1223256.1 63 21 53 5 75 16 73 2 AD-1223257.1 74 34 70 3 88 14 71 11 AD-1223258.1 68 19 73 14 91 17 104 7 AD-1223259.1 92 37 106 2 114 20 101 3 AD-1223260.1 59 12 79 8 79 16 99 13 AD-1223261.1 47 13 66 15 86 8 77 15 AD-1223262.1 39 6 45 4 68 12 58 4 AD-1223263.1 39 3 44 2 64 1 44 9 AD-1223264.1 42 10 43 3 79 20 59 8 AD-1223265.1 58 11 56 10 101 7 88 8 AD-1223266.1 49 5 48 7 68 5 72 6 AD-1223267.1 79 30 70 14 95 11 102 19 AD-1223268.1 54 7 64 7 83 9 96 6 AD-1223269.1 45 13 49 8 78 16 80 9 AD-1223270.1 53 14 70 16 76 4 70 6 AD-1223271.1 55 17 61 5 88 14 83 13 AD-1223272.1 57 28 60 6 75 9 77 6 AD-1223273.1 33 3 56 7 77 17 94 6 AD-1223274.1 49 5 67 14 81 9 110 17 AD-1223275.1 53 4 63 11 67 6 115 15 AD-1223276.1 53 3 80 15 89 6 98 9 AD-1223277.1 41 7 67 14 75 6 82 19 AD-1223278.1 47 2 60 7 69 6 58 7 AD-1223279.1 57 9 64 8 93 12 102 24 AD-1223280.1 59 8 72 2 86 11 100 9 AD-1223281.1 52 5 66 8 86 17 95 13 AD-1223282.1 35 4 56 9 67 4 103 18 AD-1223283.1 36 5 43 3 46 2 97 3 AD-1223284.1 32 1 51 9 60 15 65 7 AD-1223285.1 37 4 64 19 57 7 76 6 AD-1223286.1 37 7 38 6 51 7 56 8 AD-1223287.1 36 8 40 5 60 8 73 23 AD-1223288.1 46 6 64 10 87 5 77 6 AD-1223289.1 41 7 61 7 78 1 119 2 AD-1223290.1 46 6 56 5 76 12 98 9 AD-1223291.1 41 4 68 9 81 9 94 15 AD-1223292.1 39 10 84 36 71 4 73 9 AD-1223293.1 47 7 53 1 65 11 59 7 AD-1223294.1 38 1 53 4 59 1 61 20 AD-1223295.1 58 3 67 7 81 8 106 8 AD-1223296.1 72 1 87 10 87 14 113 19 AD-1223297.1 54 9 73 11 78 9 99 2 AD-1223298.1 55 17 65 10 75 9 94 11 AD-1223299.1 58 9 78 8 85 12 73 12 AD-1223300.1 54 6 67 7 79 20 70 9 AD-1223301.1 44 6 61 4 66 4 58 8 AD-1223302.1 44 6 67 10 77 3 75 9 AD-1223303.1 37 2 70 9 79 8 74 14 AD-1223304.1 49 3 71 8 75 9 111 13 AD-1223305.1 42 4 53 6 71 11 79 9 AD-1223306.1 39 1 60 12 64 4 71 9 AD-1223307.1 37 2 62 5 70 17 76 4 AD-1223308.1 33 1 51 12 57 15 58 6 AD-1223309.1 45 10 62 14 61 3 63 14 AD-1223310.1 41 6 54 14 62 2 62 20 AD-1223311.1 54 5 66 11 72 16 66 12 AD-1223312.1 38 7 39 2 54 8 67 19 AD-1223313.1 29 2 40 2 57 6 51 3 AD-1223314.1 33 9 43 1 47 7 58 8 AD-1223315.1 28 1 40 4 48 11 61 15

The results of the multi-dose screen in primary human hepatocytes allowed to freely uptake one set of exemplary human VEGF-A siRNAs is shown in Table 9B (correspond to siRNAs in Table 8A and 8B) The multi-dose experiments were performed at 500 nM, 100 nM, 10 nM, and 1 nM final duplex concentrations and the data are expressed as percent message remaining relative to non-targeting control.

Of the exemplary siRNA duplexes evaluated in Table 9B, 2 achieved a knockdown of VEGF-A of ≥80%, 53 achieved a knockdown of VEGF-A of ≥60%, and 239 achieved a knockdown of VEGF-A of ≥30% when administered at the 500 nM concentration.

Of the exemplary siRNA duplexes evaluated in Table 9B, 4 achieved a knockdown of VEGF-A of ≥70%, 33 achieved a knockdown of VEGF-A of ≥60%, and 235 achieved a knockdown of VEGF-A of ≥30% when administered at the 100 nM concentration.

Of the exemplary siRNA duplexes evaluated in Table 9B, 3 achieved a knockdown of VEGF-A of ≥60%, 52 achieved a knockdown of VEGF-A of ≥40%, and 113 achieved a knockdown of VEGF-A of ≥30% when administered at the 10 nM concentration.

Of the exemplary siRNA duplexes evaluated in Table 9B, 13 achieved a knockdown of VEGF-A of ≥50%, 88 achieved a knockdown of VEGF-A of ≥30%, and 146 achieved a knockdown of VEGF-A of ≥20% when administered at the 1 nM concentration.

TABLE 9B VEGF-A endogenous in vitro multi-dose screen following free uptake of one set of exemplary human VEGF-A siRNAs DuplexID 500 nM StDev 100 nM StDev 10 nM StDev 1 nM StDev AD-1222866.1 114 23 103 40 117 14 117 9 AD-1222867.1 114 11 176 58 104 9 134 24 AD-1222868.1 140 19 168 85 123 18 134 6 AD-1222869.1 110 12 149 62 149 23 131 25 AD-1222870.1 107 20 191 9 138 24 124 15 AD-1222871.1 102 11 140 48 130 13 138 18 AD-1222872.1 95 20 112 27 112 15 120 9 AD-1222873.1 73 17 94 10 107 1 110 6 AD-1222874.1 148 27 109 6 137 5 113 9 AD-1222875.1 132 7 93 4 161 25 139 11 AD-1222876.1 142 12 141 1 131 11 145 8 AD-1222877.1 119 2 111 3 176 15 132 4 AD-1222878.1 160 43 128 10 139 8 128 16 AD-1222879.1 150 21 119 13 139 22 116 16 AD-1222880.1 107 22 113 9 145 21 120 7 AD-1222881.1 90 28 91 4 106 2 113 7 AD-1222882.1 151 4 118 7 136 4 114 4 AD-1222883.1 143 13 120 11 147 12 129 8 AD-1222884.1 137 22 135 23 174 18 120 12 AD-1222885.1 162 16 130 13 169 15 120 4 AD-1222886.1 128 26 114 1 155 16 107 14 AD-1222887.1 143 32 128 24 124 10 116 13 AD-1222888.1 77 7 113 2 123 1 116 10 AD-1222889.1 142 11 92 18 143 31 113 13 AD-1222890.1 160 11 129 12 166 27 133 2 AD-1222891.1 138 12 126 6 173 27 136 17 AD-1222892.1 136 7 127 9 153 27 106 7 AD-1222893.1 111 11 118 6 141 10 130 16 AD-1222894.1 111 12 118 10 119 24 123 2 AD-1222895.1 96 8 103 0 121 27 122 18 AD-1222896.1 142 23 97 18 134 6 113 3 AD-1222897.1 155 13 126 6 157 21 129 12 AD-1222898.1 152 53 124 18 141 19 111 10 AD-1222899.1 138 21 110 9 147 28 120 1 AD-1222900.1 123 15 115 20 166 13 108 8 AD-1222901.1 106 6 116 6 161 17 105 15 AD-1222902.1 86 7 106 12 126 28 91 3 AD-1222903.1 84 5 95 2 138 6 112 23 AD-1222904.1 115 19 92 3 127 2 103 7 AD-1222905.1 137 10 129 11 132 22 107 3 AD-1222906.1 122 23 142 8 131 19 119 15 AD-1222907.1 126 35 120 11 143 22 103 3 AD-1222908.1 113 12 108 7 155 32 91 10 AD-1222909.1 104 2 106 13 134 23 106 2 AD-1222910.1 79 1 102 4 104 14 82 9 AD-1222911.1 114 8 106 22 98 11 102 6 AD-1222912.1 107 4 120 14 119 4 114 13 AD-1222913.1 126 31 115 5 109 13 103 11 AD-1222914.1 109 17 118 20 116 5 108 18 AD-1222915.1 88 3 95 11 120 21 88 14 AD-1222916.1 91 12 98 13 114 27 99 18 AD-1222917.1 70 1 93 6 88 16 86 2 AD-1222918.1 93 6 104 6 85 3 101 5 AD-1222920.1 88 12 90 10 95 6 100 8 AD-1222921.1 109 19 108 5 111 27 105 18 AD-1222922.1 91 20 87 5 124 12 104 10 AD-1222923.1 70 19 91 6 109 11 82 2 AD-1222924.1 85 14 103 13 107 14 83 17 AD-1222925.1 82 22 97 14 88 12 78 4 AD-1222926.1 103 9 86 14 92 18 93 13 AD-1222927.1 73 8 59 11 78 16 80 2 AD-1222928.1 80 1 71 11 85 9 94 7 AD-1222929.1 80 8 85 3 80 5 94 17 AD-1222930.1 76 6 85 6 97 11 74 3 AD-1222931.1 79 18 89 9 90 15 74 10 AD-1222932.1 85 1 81 16 76 3 57 7 AD-1222933.1 62 2 64 4 77 5 67 10 AD-1222934.1 43 3 45 9 63 6 62 1 AD-1222935.1 43 3 53 12 73 2 78 2 AD-1222936.1 66 11 56 11 65 10 75 6 AD-1222937.1 72 13 75 5 90 16 72 5 AD-1222938.1 65 6 71 14 78 11 81 8 AD-1222939.1 55 6 70 4 77 10 85 7 AD-1222940.1 61 16 44 1 71 6 74 2 AD-1222941.1 44 7 42 10 51 5 43 4 AD-1222942.1 31 0 37 3 64 8 59 6 AD-1222943.1 58 7 54 11 65 2 58 11 AD-1222944.1 46 6 49 6 69 7 63 12 AD-1222945.1 57 6 62 14 69 11 55 11 AD-1222946.1 50 5 60 16 55 2 57 3 AD-1222947.1 37 4 43 9 59 5 62 9 AD-1222948.1 60 1 63 16 61 13 57 1 AD-1222949.1 40 7 41 12 61 7 46 1 AD-1222950.1 36 4 36 7 55 1 44 4 AD-1222951.1 34 5 32 6 52 4 41 5 AD-1222952.1 61 6 48 14 65 7 44 8 AD-1222953.1 34 6 30 5 51 6 43 1 AD-1222954.1 15 2 25 5 47 4 36 1 AD-1222955.1 19 3 26 5 52 8 40 1 AD-1222956.1 64 8 72 5 110 6 123 6 AD-1222957.1 132 19 98 5 109 14 122 3 AD-1222958.1 72 10 96 7 120 26 133 3 AD-1222959.1 76 13 88 2 119 29 125 4 AD-1222960.1 71 3 123 14 136 39 141 8 AD-1222961.1 112 10 119 10 128 41 115 1 AD-1222962.1 88 4 97 16 120 31 142 12 AD-1222963.1 81 13 83 7 129 23 123 2 AD-1222964.1 90 6 89 2 130 39 134 10 AD-1222965.1 141 16 118 6 118 10 145 19 AD-1222966.1 105 8 119 9 113 11 145 20 AD-1222967.1 127 12 126 3 116 6 143 6 AD-1222968.1 128 7 144 9 120 19 138 11 AD-1222969.1 114 12 121 17 110 6 126 7 AD-1222970.1 58 6 92 7 104 10 120 5 AD-1222971.1 81 6 109 9 106 7 127 8 AD-1222972.1 95 21 76 5 100 11 119 12 AD-1222973.1 97 12 87 2 101 34 143 1 AD-1222974.1 119 8 122 8 105 11 116 5 AD-1222975.1 115 11 113 8 102 9 130 10 AD-1222976.1 97 22 100 13 91 8 106 11 AD-1222977.1 90 13 86 12 98 5 109 4 AD-1222978.1 66 7 77 2 104 12 136 1 AD-1222979.1 92 5 93 4 98 1 125 4 AD-1222980.1 98 1 117 12 103 3 133 9 AD-1222981.1 147 17 147 19 110 10 119 10 AD-1222982.1 95 10 99 16 92 10 114 6 AD-1222983.1 77 9 86 14 86 3 108 10 AD-1222984.1 73 15 93 5 78 3 108 10 AD-1222985.1 89 12 103 20 101 22 119 10 AD-1222986.1 65 1 75 1 97 8 120 10 AD-1222987.1 105 10 92 6 106 9 122 6 AD-1222988.1 74 0 79 1 90 11 128 7 AD-1222989.1 116 1 99 7 112 29 114 11 AD-1222990.1 87 1 95 8 97 15 116 7 AD-1222991.1 81 11 95 14 100 10 109 6 AD-1222992.1 78 10 100 5 89 7 109 6 AD-1222993.1 78 3 111 9 105 10 124 7 AD-1222994.1 78 12 76 0 99 13 116 16 AD-1222995.1 103 18 93 0 97 6 120 3 AD-1222996.1 108 4 122 17 104 5 114 11 AD-1222997.1 108 12 96 3 94 8 113 15 AD-1222998.1 101 11 77 3 87 7 106 2 AD-1222999.1 94 8 99 8 116 8 105 5 AD-1223000.1 80 14 97 25 80 10 97 8 AD-1223001.1 79 10 84 8 108 17 111 5 AD-1223002.1 95 15 93 12 109 11 120 16 AD-1223003.1 97 9 105 9 105 10 115 8 AD-1223004.1 64 9 80 9 93 16 101 10 AD-1223005.1 85 11 104 1 89 16 93 11 AD-1223006.1 70 1 83 9 82 5 96 1 AD-1223007.1 76 2 86 15 88 20 100 8 AD-1223008.1 37 3 66 9 88 2 110 2 AD-1223009.1 57 9 73 10 83 9 102 4 AD-1223010.1 73 7 82 6 108 19 109 9 AD-1223011.1 71 2 86 16 89 15 107 7 AD-1223012.1 71 10 92 14 96 11 99 6 AD-1223013.1 99 9 114 19 79 23 100 5 AD-1223014.1 77 4 95 10 87 4 101 5 AD-1223015.1 69 13 86 8 82 14 97 12 AD-1223016.1 52 1 76 7 95 18 101 11 AD-1223017.1 45 6 55 1 67 7 84 8 AD-1223018.1 39 9 60 2 80 9 90 4 AD-1223019.1 62 6 59 11 87 6 86 4 AD-1223020.1 60 13 65 2 68 28 82 13 AD-1223021.1 76 6 73 19 70 10 85 8 AD-1223022.1 60 10 64 3 70 3 77 5 AD-1223023.1 56 4 62 3 90 24 73 7 AD-1223024.1 52 3 60 3 63 5 71 21 AD-1223025.1 53 10 64 3 76 3 86 10 AD-1223026.1 63 5 53 7 62 15 85 10 AD-1223027.1 72 14 79 14 80 10 83 4 AD-1223028.1 64 7 68 7 74 3 81 11 AD-1223029.1 78 4 75 17 68 11 82 6 AD-1223030.1 41 8 49 6 68 4 69 7 AD-1223031.1 26 1 34 1 37 6 48 4 AD-1223032.1 35 1 49 5 56 8 73 1 AD-1223033.1 34 6 43 3 59 13 71 3 AD-1223034.1 49 5 50 1 68 2 82 6 AD-1223035.1 36 3 42 8 55 1 74 8 AD-1223036.1 32 5 39 11 49 2 75 5 AD-1223037.1 28 8 29 4 47 11 68 6 AD-1223038.1 23 4 38 6 49 5 69 5 AD-1223039.1 26 9 40 1 41 4 61 11 AD-1223040.1 29 5 37 6 37 4 54 3 AD-1223041.1 31 1 35 2 41 1 53 6 AD-1223042.1 29 4 32 5 42 12 64 10 AD-1223043.1 27 3 40 7 47 6 58 4 AD-1223044.1 29 2 40 3 39 1 55 1 AD-1223045.1 31 2 44 3 42 8 62 5 AD-1223046.1 39 2 42 3 69 4 92 15 AD-1223047.1 35 8 38 5 69 5 80 7 AD-1223048.1 30 2 40 4 71 4 92 21 AD-1223049.1 38 1 45 11 65 2 86 16 AD-1223050.1 36 5 51 6 67 3 82 17 AD-1223051.1 47 7 56 7 68 3 66 10 AD-1223052.1 39 12 47 2 68 1 66 7 AD-1223053.1 42 4 58 15 63 1 56 6 AD-1223054.1 43 9 53 5 87 2 88 1 AD-1223055.1 38 10 52 10 84 6 93 2 AD-1223056.1 49 17 51 9 80 6 125 26 AD-1223057.1 50 3 45 11 81 3 95 12 AD-1223058.1 29 6 38 8 77 4 92 27 AD-1223059.1 28 2 37 6 77 7 67 15 AD-1223060.1 33 1 51 10 68 3 58 1 AD-1223061.1 31 1 44 4 67 3 68 13 AD-1223062.1 57 10 59 4 103 8 80 13 AD-1223063.1 66 5 57 5 102 6 98 24 AD-1223064.1 65 16 75 11 109 14 91 1 AD-1223065.1 50 2 67 22 103 2 91 19 AD-1223066.1 30 5 37 4 84 6 76 1 AD-1223067.1 40 3 54 12 81 9 71 11 AD-1223068.1 36 1 37 5 70 6 61 6 AD-1223069.1 67 27 73 11 99 17 103 21 AD-1223070.1 65 19 58 3 105 3 116 24 AD-1223071.1 40 5 58 14 96 8 90 2 AD-1223072.1 65 14 85 24 115 15 74 15 AD-1223073.1 39 1 59 9 110 14 79 17 AD-1223074.1 42 6 54 5 87 4 63 5 AD-1223075.1 47 10 38 2 78 3 54 2 AD-1223076.1 78 20 70 16 68 14 99 22 AD-1223077.1 70 14 76 11 103 19 90 5 AD-1223078.1 105 6 113 26 106 9 122 9 AD-1223079.1 64 11 76 4 107 7 98 16 AD-1223080.1 79 10 67 19 87 4 89 19 AD-1223081.1 67 21 80 17 99 5 82 16 AD-1223082.1 37 4 45 16 85 13 68 15 AD-1223083.1 44 16 50 7 78 6 64 12 AD-1223084.1 59 12 57 1 96 17 95 3 AD-1223085.1 86 28 79 8 100 3 101 15 AD-1223086.1 67 8 58 6 93 9 123 3 AD-1223087.1 82 21 101 5 95 13 97 8 AD-1223088.1 76 25 92 2 127 20 100 21 AD-1223089.1 78 17 68 17 93 2 83 17 AD-1223090.1 38 3 55 13 84 10 63 16 AD-1223091.1 78 15 60 9 97 23 113 3 AD-1223092.1 66 3 69 16 112 13 79 18 AD-1223093.1 65 1 60 5 119 23 95 15 AD-1223094.1 57 10 61 11 92 7 87 11 AD-1223095.1 67 2 60 9 88 11 97 4 AD-1223096.1 52 20 57 5 100 23 66 13 AD-1223097.1 40 2 64 8 80 7 57 4 AD-1223098.1 41 13 47 6 72 2 50 6 AD-1223099.1 60 18 66 5 76 17 103 20 AD-1223100.1 46 15 54 10 77 6 81 4 AD-1223101.1 76 8 67 16 110 15 91 17 AD-1223102.1 45 6 69 13 96 15 90 6 AD-1223103.1 70 17 71 2 107 18 65 1 AD-1223104.1 57 22 52 8 73 9 55 4 AD-1223105.1 48 15 69 16 84 25 64 16 AD-1223106.1 61 8 59 13 95 6 53 4 AD-1223107.1 71 2 60 20 77 9 89 11 AD-1223108.1 78 3 62 5 85 13 78 5 AD-1223109.1 74 18 63 18 85 0 84 19 AD-1223110.1 64 4 78 9 104 29 73 10 AD-1223111.1 90 8 74 19 83 21 72 15 AD-1223112.1 71 10 87 14 78 14 66 8 AD-1223113.1 62 12 80 1 83 20 60 5 AD-1223114.1 71 18 62 9 68 1 62 10 AD-1223115.1 111 5 80 15 82 7 82 3 AD-1223116.1 80 22 80 10 73 7 92 2 AD-1223117.1 84 23 98 2 92 23 73 17 AD-1223118.1 71 25 67 17 82 26 63 5 AD-1223119.1 66 15 91 33 82 31 87 21 AD-1223120.1 61 6 64 13 88 7 59 4 AD-1223121.1 66 9 49 11 66 5 59 6 AD-1223122.1 90 11 68 20 62 10 74 15 AD-1223123.1 61 22 52 20 53 0 63 4 AD-1223124.1 89 12 72 16 65 5 83 11 AD-1223125.1 77 22 72 18 65 7 73 8 AD-1223126.1 96 18 80 20 77 32 74 13 AD-1223127.1 54 11 60 9 53 9 64 12 AD-1223128.1 58 16 45 1 67 1 65 8 AD-1223129.1 45 7 39 6 56 5 60 1 AD-1223130.1 37 2 50 3 51 5 74 7 AD-1223131.1 57 17 45 13 66 5 70 2 AD-1223132.1 72 16 53 5 71 18 92 7 AD-1223133.1 42 3 57 7 54 2 61 9 AD-1223134.1 49 11 46 1 59 1 84 22 AD-1223135.1 46 3 50 11 57 5 69 1 AD-1223136.1 101 19 100 15 88 26 159 78 AD-1223137.1 88 14 92 13 98 19 91 23 AD-1223138.1 75 9 67 4 71 17 86 26 AD-1223139.1 89 19 63 6 97 31 82 10 AD-1223140.1 90 28 71 8 59 10 76 12 AD-1223141.1 83 3 64 9 62 6 75 14 AD-1223142.1 102 5 61 1 74 19 73 2 AD-1223143.1 96 31 54 1 57 15 88 15 AD-1223144.1 91 16 82 10 112 9 129 35 AD-1223145.1 86 15 74 10 86 5 125 21 AD-1223146.1 81 10 85 3 94 1 143 21 AD-1223147.1 99 15 80 17 86 10 109 24 AD-1223148.1 93 22 82 12 91 17 115 18 AD-1223149.1 88 6 74 4 88 15 110 11 AD-1223150.1 79 6 66 1 71 5 103 17 AD-1223151.1 98 20 64 9 61 17 108 20 AD-1223152.1 121 28 92 14 78 5 102 17 AD-1223153.1 80 15 93 9 105 7 122 23 AD-1223154.1 73 14 85 5 87 13 109 10 AD-1223155.1 87 2 68 3 92 12 108 17 AD-1223156.1 74 19 72 2 73 8 102 12 AD-1223157.1 83 21 76 5 78 12 101 20 AD-1223158.1 78 22 64 12 75 2 97 24 AD-1223159.1 102 36 75 9 115 22 104 16 AD-1223160.1 116 20 122 15 105 13 154 18 AD-1223161.1 80 14 96 7 114 14 99 6 AD-1223162.1 60 5 76 7 97 18 111 24 AD-1223163.1 85 18 79 10 83 15 118 22 AD-1223164.1 75 1 95 21 78 8 113 7 AD-1223165.1 73 7 68 8 66 2 111 28 AD-1223166.1 106 19 92 4 98 37 100 11 AD-1223167.1 111 12 116 21 121 5 124 11 AD-1223168.1 102 20 93 2 124 25 145 4 AD-1223169.1 93 23 89 18 132 17 133 5 AD-1223170.1 85 14 79 25 111 11 119 26 AD-1223171.1 82 19 77 9 89 12 121 30 AD-1223172.1 90 3 76 5 92 7 89 6 AD-1223173.1 74 14 79 15 85 18 119 36 AD-1223174.1 135 52 80 8 90 3 95 15 AD-1223175.1 86 20 96 8 87 4 116 5 AD-1223176.1 88 17 104 12 99 15 123 3 AD-1223177.1 81 14 78 6 90 8 109 23 AD-1223178.1 106 7 80 14 88 2 118 15 AD-1223179.1 83 14 71 10 93 8 95 18 AD-1223180.1 61 19 67 9 79 9 127 4 AD-1223181.1 105 31 80 5 99 12 106 12 AD-1223182.1 69 2 81 12 88 5 105 20 AD-1223183.1 108 38 85 6 106 9 123 17 AD-1223184.1 45 6 61 4 75 8 82 12 AD-1223185.1 59 4 76 15 75 3 107 20 AD-1223186.1 66 12 62 10 88 11 89 7 AD-1223187.1 63 10 58 10 72 7 99 20 AD-1223188.1 51 12 47 8 71 9 90 19 AD-1223189.1 73 15 56 3 75 11 85 2 AD-1223190.1 57 13 61 5 80 13 89 18 AD-1223191.1 55 14 68 9 71 1 100 8 AD-1223192.1 39 3 61 9 73 4 92 16 AD-1223193.1 41 9 51 2 72 9 69 4 AD-1223194.1 34 2 44 5 61 9 79 18 AD-1223195.1 52 17 38 5 65 11 89 12 AD-1223196.1 36 4 39 4 89 22 85 20 AD-1223197.1 66 13 40 6 62 16 70 10 AD-1223198.1 56 8 66 5 78 23 95 14 AD-1223199.1 56 17 61 6 75 5 113 30 AD-1223200.1 51 14 51 9 68 14 102 17 AD-1223201.1 60 15 53 3 59 6 81 3 AD-1223202.1 36 10 46 3 57 9 95 1 AD-1223203.1 43 13 34 1 66 13 82 19 AD-1223204.1 63 16 49 3 60 5 57 3 AD-1223205.1 51 14 57 5 75 3 75 12 AD-1223206.1 30 4 49 10 69 14 67 6 AD-1223207.1 67 11 67 6 67 7 77 11 AD-1223208.1 64 11 61 8 68 4 84 24 AD-1223209.1 56 12 62 7 73 11 86 16 AD-1223210.1 58 33 40 2 69 8 93 2 AD-1223211.1 39 0 48 4 52 15 57 5 AD-1223212.1 65 5 57 2 53 2 95 20 AD-1223213.1 55 11 57 3 69 5 78 10 AD-1223214.1 28 2 46 1 53 8 70 5 AD-1223215.1 37 2 52 1 61 12 80 11 AD-1223216.1 42 14 47 3 67 31 72 6 AD-1223217.1 43 7 52 3 66 5 86 2 AD-1223218.1 64 26 45 6 59 13 86 11 AD-1223219.1 70 10 55 7 56 8 58 3 AD-1223220.1 54 15 64 3 42 4 57 2 AD-1223221.1 51 15 55 6 45 8 62 8 AD-1223222.1 70 5 66 5 58 6 86 20 AD-1223223.1 37 5 54 4 59 14 61 15 AD-1223224.1 55 4 40 1 51 4 61 3 AD-1223225.1 49 17 41 6 61 11 68 1 AD-1223226.1 57 3 46 12 71 4 83 19 AD-1223227.1 73 7 55 1 80 7 92 14 AD-1223228.1 51 5 41 3 72 4 89 6 AD-1223229.1 63 7 54 11 86 5 99 13 AD-1223230.1 52 7 46 8 81 8 81 12 AD-1223231.1 61 4 52 1 78 9 88 11 AD-1223232.1 76 6 64 7 75 8 80 13 AD-1223233.1 70 8 53 6 77 5 66 12 AD-1223234.1 68 11 56 14 81 5 94 14 AD-1223235.1 58 4 41 2 94 2 96 7 AD-1223236.1 72 4 62 19 101 10 99 6 AD-1223237.1 78 10 63 3 109 5 103 20 AD-1223238.1 67 16 65 22 102 14 100 15 AD-1223239.1 58 2 57 3 90 8 89 21 AD-1223240.1 57 1 53 8 87 7 79 12 AD-1223241.1 62 9 50 10 73 11 70 18 AD-1223242.1 54 9 55 11 70 8 87 4 AD-1223243.1 62 11 48 4 77 6 114 6 AD-1223244.1 57 5 54 9 94 9 105 18 AD-1223245.1 75 8 78 2 111 28 116 9 AD-1223246.1 51 4 49 3 85 8 92 6 AD-1223247.1 61 7 53 11 87 10 81 6 AD-1223248.1 60 2 54 11 75 1 72 11 AD-1223249.1 65 7 71 5 85 12 111 8 AD-1223250.1 85 15 89 3 118 14 131 1 AD-1223251.1 74 6 88 5 108 8 102 2 AD-1223252.1 55 3 78 19 107 19 95 11 AD-1223253.1 48 7 69 6 94 9 90 13 AD-1223254.1 58 11 59 21 96 15 83 5 AD-1223255.1 48 5 58 11 79 5 68 2 AD-1223256.1 51 9 53 7 75 4 99 11 AD-1223257.1 44 2 43 6 98 10 120 15 AD-1223258.1 68 8 67 6 107 12 111 6 AD-1223259.1 96 7 103 8 111 15 106 1 AD-1223260.1 82 2 80 7 114 15 112 11 AD-1223261.1 62 1 64 3 99 20 95 1 AD-1223262.1 48 10 48 9 86 8 90 6 AD-1223263.1 51 5 49 6 71 3 71 5 AD-1223264.1 51 2 56 13 75 7 79 3 AD-1223265.1 48 3 47 1 82 1 94 12 AD-1223266.1 42 6 49 5 84 1 105 9 AD-1223267.1 76 2 101 20 105 7 108 11 AD-1223268.1 63 12 83 28 96 13 86 17 AD-1223269.1 43 4 51 17 93 0 84 10 AD-1223270.1 63 3 56 7 96 6 95 1 AD-1223271.1 58 1 63 18 71 2 93 11 AD-1223272.1 55 7 56 11 82 1 106 7 AD-1223273.1 66 10 63 18 106 9 120 15 AD-1223274.1 67 6 64 13 97 13 98 12 AD-1223275.1 67 8 88 17 103 9 79 8 AD-1223276.1 75 10 64 13 94 13 84 3 AD-1223277.1 73 5 62 6 88 15 82 1 AD-1223278.1 60 3 61 7 83 10 81 5 AD-1223279.1 65 3 64 12 88 12 93 13 AD-1223280.1 67 2 67 9 89 4 119 9 AD-1223281.1 68 8 62 16 95 10 96 2 AD-1223282.1 57 7 73 2 87 8 95 3 AD-1223283.1 50 3 75 3 75 2 80 1 AD-1223284.1 51 7 61 26 84 9 80 2 AD-1223285.1 70 13 70 10 89 15 87 8 AD-1223286.1 43 1 48 8 73 18 77 8 AD-1223287.1 50 4 46 16 65 4 76 0 AD-1223288.1 56 9 55 20 75 10 78 3 AD-1223289.1 72 19 57 10 87 3 91 10 AD-1223290.1 67 2 73 5 88 13 89 6 AD-1223291.1 74 11 69 15 81 1 70 8 AD-1223292.1 61 2 58 5 81 2 78 1 AD-1223293.1 63 9 60 6 82 4 74 6 AD-1223294.1 54 2 56 10 53 2 68 1 AD-1223295.1 80 14 90 3 75 10 76 13 AD-1223296.1 92 20 109 8 87 10 95 11 AD-1223297.1 75 9 89 2 80 5 96 4 AD-1223298.1 75 6 79 10 86 11 94 2 AD-1223299.1 77 8 79 6 89 6 82 1 AD-1223300.1 78 15 72 5 76 5 71 8 AD-1223301.1 64 4 67 9 57 2 60 8 AD-1223302.1 66 5 79 17 66 4 71 9 AD-1223303.1 78 9 86 14 68 1 74 9 AD-1223304.1 64 2 76 10 72 7 72 5 AD-1223305.1 59 4 72 11 66 3 78 13 AD-1223306.1 58 1 51 33 77 23 81 13 AD-1223307.1 61 9 78 15 63 4 70 5 AD-1223308.1 52 7 52 10 63 5 58 6 AD-1223309.1 60 1 39 4 59 4 60 5 AD-1223310.1 52 5 40 7 60 2 55 4 AD-1223311.1 62 7 59 13 62 2 54 5 AD-1223312.1 49 8 44 8 50 5 56 8 AD-1223313.1 43 5 44 9 51 3 50 0 AD-1223314.1 45 8 39 4 53 5 49 6 AD-1223315.1 53 4 46 7 56 5 50 6

The results of the multi-dose screen in primary human hepatocytes transfected with an additional set of exemplary human VEGF-A siRNAs is shown in Table 11 (correspond to modified siRNAs in Table 10A). The multi-dose experiments were performed at 50 nM, 10 nM, 1 nM, and 0.1 nM final duplex concentrations and the data are expressed as percent message remaining relative to non-targeting control.

Of the exemplary siRNA duplexes evaluated in Table 11, 6 achieved a knockdown of VEGF-A of ≥70%, 34 achieved a knockdown of VEGF-A of ≥60%, 49 achieved a knockdown of VEGF-A of ≥50%, 62 achieved a knockdown of VEGF-A of ≥30%, and 75 achieved a knockdown of VEGF-A of ≥20% when administered at the 50 nM concentration.

Of the exemplary siRNA duplexes evaluated in Table 11, 2 achieved a knockdown of VEGF-A of ≥70%, 18 achieved a knockdown of VEGF-A of ≥60%, 35 achieved a knockdown of VEGF-A of ≥50%, 66 achieved a knockdown of VEGF-A of ≥30%, and 77 achieved a knockdown of VEGF-A of ≥20% when administered at the 10 nM concentration.

Of the exemplary siRNA duplexes evaluated in Table 11, 13 achieved a knockdown of VEGF-A of ≥50%, 33 achieved a knockdown of VEGF-A of ≥40%, 49 achieved a knockdown of VEGF-A of ≥30%, 62 achieved a knockdown of VEGF-A of ≥20%, and 74 achieved a knockdown of VEGF-A of ≥10% when administered at the 1 nM concentration.

Of the exemplary siRNA duplexes evaluated in Table 11, 2 achieved a knockdown of VEGF-A of ≥40%, 7 achieved a knockdown of VEGF-A of ≥30%, 25 achieved a knockdown of VEGF-A of ≥20%, 46 achieved a knockdown of VEGF-A of ≥10%, and 55 achieved a knockdown of VEGF-A of ≥5% when administered at the 0.1 nM concentration.

TABLE 11 VEGF-A endogenous in vitro multi-dose screen following cellular transfection with additional set of exemplary human VEGF-A siRNAs 50 nM dose 10 nM dose 1 nM dose 0.1 nM dose Duplex Avg SD Avg SD Avg SD Avg SD AD-1353514.1 73.2 14.7 47.83 6.33 89.52 8.82 108.56 17.50 AD-1353484.1 60.9 2.1 30.99 3.11 46.98 8.32 74.34 10.43 AD-1353454.1 32.1 2.1 31.33 5.04 48.05 7.92 52.74 3.79 AD-1353468.1 36.5 10.1 37.70 11.20 54.44 8.95 76.93 5.45 AD-1353498.1 71.4 8.9 70.68 17.79 91.31 15.16 119.43 2.90 AD-1353438.1 30.4 5.0 36.17 11.97 49.92 9.56 68.61 10.47 AD-1353515.1 85.4 4.7 59.45 15.56 86.89 15.76 119.70 10.55 AD-1353485.1 61.2 3.6 67.37 28.56 73.00 19.46 101.36 12.30 AD-1353455.1 37.7 5.8 39.73 4.39 57.46 11.43 75.26 9.60 AD-1353513.1 72.4 19.2 70.36 7.66 65.81 2.00 99.31 4.26 AD-1353483.1 45.4 7.9 59.48 13.52 63.00 1.14 88.59 7.06 AD-1353453.1 49.4 3.1 52.47 11.81 73.02 14.48 87.42 1.86 AD-1353502.1 111.8 3.9 122.89 9.45 108.63 2.65 101.24 12.32 AD-1353472.1 77.7 1.1 77.95 21.21 82.89 10.59 110.13 1.33 AD-1353442.1 43.1 5.9 58.75 26.58 53.92 6.62 86.50 13.94 AD-1353499.1 52.6 8.5 50.86 1.41 60.86 4.25 68.29 32.87 AD-1353469.1 34.2 4.9 26.91 8.25 52.31 12.97 68.94 3.00 AD-1353439.1 40.5 7.0 33.92 5.01 47.72 5.18 59.98 0.64 AD-1353516.1 73.4 2.3 91.54 6.70 96.22 13.84 109.69 16.01 AD-1353486.1 55.2 10.8 69.54 22.45 66.89 15.26 85.60 12.44 AD-1353456.1 36.7 2.3 40.99 2.33 47.06 0.75 77.80 12.27 AD-1353509.1 78.2 15.5 65.05 19.24 79.91 15.99 92.15 6.76 AD-1353479.1 34.1 1.7 59.07 11.65 61.37 12.56 81.86 11.12 AD-1353449.1 37.2 3.1 43.76 2.00 64.46 14.54 94.21 15.51 AD-1353503.1 107.2 12.2 124.19 25.48 129.99 12.55 103.32 5.51 AD-1353473.1 69.5 12.8 81.70 4.74 73.09 4.34 96.57 7.16 AD-1353443.1 56.0 5.3 47.76 1.37 60.14 2.14 78.45 2.90 AD-1353506.1 125.4 15.1 72.10 10.55 98.53 31.92 139.95 25.71 AD-1353476.1 31.1 0.9 52.33 18.02 53.46 8.06 81.81 20.46 AD-1353446.1 33.5 3.9 49.43 19.14 48.57 12.81 89.09 18.37 AD-1353497.1 73.7 15.2 76.17 18.94 110.55 19.54 108.38 14.24 AD-1353467.1 24.1 1.6 68.83 36.91 60.52 9.05 88.11 6.79 AD-1353437.1 26.6 1.3 36.54 18.99 58.73 11.11 78.39 13.30 AD-1353494.1 78.9 18.5 64.78 5.56 80.76 28.61 105.16 8.40 AD-1353464.1 39.2 11.1 43.51 10.28 46.64 1.22 86.15 13.43 AD-1353434.1 25.8 2.3 35.87 7.53 46.49 2.58 88.48 12.93 AD-1353505.1 79.3 14.0 90.37 26.82 88.87 9.99 118.05 27.89 AD-1353475.1 45.8 4.4 81.74 24.42 61.65 9.73 113.65 24.35 AD-1353445.1 33.1 4.6 34.12 2.32 58.71 12.42 76.70 5.84 AD-1353518.1 106.7 9.5 66.77 10.60 101.34 19.91 116.14 13.23 AD-1353490.1 57.6 2.9 65.55 2.51 59.72 1.89 82.32 10.94 AD-1353460.1 43.3 2.2 57.20 17.51 57.83 6.41 81.60 3.42 AD-1353512.1 49.3 2.8 56.05 16.30 73.12 5.01 80.00 12.61 AD-1353482.1 31.8 7.0 41.02 4.08 49.87 13.58 75.30 11.76 AD-1353452.1 22.0 0.8 27.69 6.99 49.18 8.36 64.25 8.77 AD-1353501.1 93.4 14.4 73.05 23.54 91.52 22.64 118.07 3.73 AD-1353471.1 35.3 16.6 55.66 10.36 62.03 8.43 74.30 10.30 AD-1353441.1 42.8 12.1 37.07 2.05 55.05 11.83 87.87 11.35 AD-1353495.1 64.9 6.6 57.36 3.08 76.11 7.59 91.83 2.54 AD-1353465.1 46.1 9.0 52.06 2.82 71.33 9.34 96.77 3.94 AD-1353435.1 26.6 0.6 34.92 2.32 52.83 10.26 75.24 2.38 AD-1353510.1 119.8 18.1 101.24 29.18 93.46 10.24 113.16 12.92 AD-1353480.1 31.4 6.3 39.32 0.42 41.34 14.17 67.02 5.59 AD-1353450.1 34.6 7.4 66.32 23.95 50.20 0.35 73.85 1.29 AD-1353492.1 77.7 10.8 71.56 11.36 77.86 8.02 121.84 4.81 AD-1353462.1 77.9 8.7 61.05 3.21 84.55 11.04 95.78 4.27 AD-1353432.1 53.3 2.4 54.18 15.15 77.02 10.61 91.59 3.58 AD-1353504.1 112.3 16.9 103.54 22.58 96.95 21.25 104.68 1.67 AD-1353474.1 37.3 0.8 45.61 7.93 64.86 13.55 90.57 9.24 AD-1353444.1 34.6 2.7 46.49 12.28 64.50 3.69 90.43 15.39 AD-1353493.1 115.2 3.5 109.69 24.35 115.11 15.22 148.20 6.03 AD-1353463.1 37.4 10.6 39.98 1.58 67.51 13.18 101.55 10.32 AD-1353433.1 41.6 2.6 49.27 11.07 57.69 7.11 85.23 1.77 AD-1353508.1 99.1 18.5 108.35 7.54 119.28 22.06 127.04 26.81 AD-1353478.1 61.3 7.9 80.29 6.74 84.40 5.07 106.24 18.11 AD-1353448.1 46.8 10.5 45.28 0.83 70.79 13.97 93.49 2.61 AD-1353496.1 71.4 11.4 72.67 26.10 75.08 10.38 78.24 0.83 AD-1353466.1 27.1 2.2 41.23 4.27 46.93 8.96 74.57 10.13 AD-1353436.1 34.3 12.7 44.21 16.99 76.15 17.23 100.46 13.86 AD-1353500.1 93.3 10.4 71.79 21.42 90.97 13.97 121.98 8.36 AD-1353470.1 56.5 18.5 68.60 21.85 66.08 16.35 98.20 16.07 AD-1353440.1 42.7 13.9 32.74 2.42 47.72 4.01 87.22 13.60 AD-1334067.3 62.0 5.0 66.03 15.65 104.31 6.24 118.77 15.26 AD-1353488.1 45.4 9.5 54.95 18.87 66.30 5.64 93.92 6.82 AD-1353458.1 49.9 2.7 42.68 4.29 52.79 7.82 86.26 5.91 AD-1334065.3 71.1 12.6 78.09 22.23 84.66 18.18 114.25 13.96 AD-1353487.1 34.9 4.5 66.90 18.73 56.96 8.69 78.72 9.61 AD-1353457.1 38.2 9.3 58.10 28.97 54.23 13.92 72.52 6.80 AD-1353511.1 83.9 11.7 62.66 20.11 82.39 4.29 108.94 17.57 AD-1353481.1 37.4 5.3 33.15 14.70 53.45 6.54 89.49 20.18 AD-1353451.1 36.5 3.0 50.19 3.34 70.69 2.05 89.11 5.24 AD-1353507.1 88.8 24.1 103.80 3.43 118.16 22.78 109.81 8.53 AD-1353477.1 58.1 13.7 71.67 24.75 89.22 12.01 97.98 5.38 AD-1353447.1 35.9 5.8 35.44 0.37 69.83 7.21 93.70 5.60 AD-1353517.1 81.4 27.8 65.25 15.18 87.98 15.64 87.33 5.55 AD-1353489.1 39.1 8.5 48.80 14.60 53.72 2.50 78.25 4.63 AD-1353459.1 36.3 3.0 42.48 5.66 55.07 0.79 88.61 15.57 AD-1353519.1 99.4 27.7 81.48 9.81 93.30 21.44 129.30 24.38 AD-1353491.1 49.6 22.3 68.94 16.28 84.63 0.34 82.41 4.90 AD-1353461.1 36.0 7.6 42.22 11.83 52.71 11.40 75.56 6.49

Example 3. In Vivo Screening of VEGF-A siRNA

This Example investigates the effects of the exemplary VEGF-A targeting siRNAs for in vivo efficacy for human VEGF-A knockdown in AAV mice. The first exemplary set of VEGF-A targeting siRNAs investigated includes AD-64228, AD-953374, AD-953504, AD-953336, AD-953337, AD-901376, AD-953364, AD-953340, AD-953351, AD-953342, AD-953308, AD-953344, AD-953339, and AD-953363 (summarized in Table 12 and FIGS. 1A-1B). The second set of exemplary VEGF-A targeting siRNAs investigated included AD-901349, AD-953481, AD-901356, AD-901355, AD-953365, AD-953410, AD-953411, AD-953338, AD-953350, AD-953375, AD-953341, AD-953370, AD-953386, AD-64958 (summarized in Table 13 and FIGS. 3A-3B) The final set of exemplary VEGF-A targeting siRNAs investigated included AD-1397050, AD-1397051, AD-1397052, AD-1397053, AD-1397054, AD-1397055, AD-1397056, AD-1397058, AD-1397059, AD-1397060, AD-1397061, AD-1397062, AD-1397064, AD-1397065, AD-1397066, AD-1397067, AD-1397068, AD-1397069, and AD-64958 (summarized in Table 14 and FIGS. 5A-5C).

TABLE 12 VEGF-A in vivo single-dose screen with one set of exemplary VEGF-A siRNA duplexes. In this table the column “Duplex Name” provides the numerical part of the duplex name. The duplex name can comprise a suffix (number following the decimal point in a duplex name) that merely refers to a batch  production number. The suffix can be omitted from the duplex name without changing the chemical structure. For example, duplex AD-953504.1 in Table 4A refers to the same duplex as AD-953504 in Table 12. Duplex SEQ ID Name Strand Target Modified Sequence (5′-3′) NO AD-64228 sense None asascaguGfuUfCfUfugcucuauaaL96 4162 anti- mTTR usUfsauaGfaGfCfaagaAfcAfcuguususu 4163 sense AD-953504 sense VEGF-A asasaau(Ahd)gadCadTugcuauucuaL96 1037 anti- VEGF-A VPusdAsgadAudAgcaadTgdTcdTauuuusasu 1167 sense AD-953308 sense VEGF-A csascca(Uhd)GfcAfGfAfuuaugcggaaL96  579 anti- VEGF-A VPusUfsccgCfaUfAfaucuGfcAfuggugsasu  709 sense AD-953336 sense VEGF-A asasaga(Chd)UfgAfUfAfcagaacgauaL96  518 anti- VEGF-A VPusAfsucgUfuCfUfguauCfaGfucuuuscsc  648 sense AD-953337 sense VEGF-A asasgac(Uhd)GfaUfAfCfagaacgaucaL96  522 anti- VEGF-A VPusGfsaucGfuUfCfuguaUfcAfgucuususc  652 sense AD-953339 sense VEGF-A gsascug(Ahd)UfaCfAfGfaacgaucgaaL96  528 anti- VEGF-A VPusUfscgaUfcGfUfucugUfaUfcagucsusu  658 sense AD-953340 sense VEGF-A ascsuga(Uhd)AfcAfGfAfacgaucgauaL96  517 anti- VEGF-A VPusAfsucgAfuCfGfuucuGfuAfucaguscsu  647 sense AD-953342 sense VEGF-A asusaca(Ghd)AfaCfGfAfucgauacagaL96  523 anti- VEGF-A VPusCfsuguAfuCfGfaucgUfuCfuguauscsa  653 sense AD-953344 sense VEGF-A csasgaa(Chd)AfgUfCfCfuuaauccagaL96  527 anti- VEGF-A VPusCfsuggAfuUfAfaggaCfuGfuucugsusc  657 sense AD-953351 sense VEGF-A asgsugc(Uhd)AfaUfGfUfuauugguguaL96  540 anti- VEGF-A VPusAfscacCfaAfUfaacaUfuAfgcacusgsu  670 sense AD-953363 sense VEGF-A gsasgaa(Ahd)GfuGfUfUfuuauauacgaL96  519 anti- VEGF-A VPusCfsguaUfaUfAfaaacAfcUfuucucsusu  649 sense AD-953364 sense VEGF-A ascsggu(Ahd)CfuUfAfUfuuaauauccaL96  567 anti- VEGF-A VPusGfsgauAfuUfAfaauaAfgUfaccgusasu  697 sense AD-901376 sense VEGF-A ascsggu(Ahd)CfuUfAfUfuuaauauccaL96 4157 anti- VEGF-A VPusGfsgaua(Tgn)uaaauaAfgUfaccgusasu  131 sense AD-953374 sense VEGF-A asasaau(Ahd)GfaCfAfUfugcuauucuaL96  553 anti- VEGF-A VPusAfsgaaUfaGfCfaaugUfcUfauuuusasu  683 sense

TABLE 13 VEGF-A in vivo single-dose screen with one set of exemplary VEGF-A siRNA duplexes. In this table the column “Duplex Name” provides the numerical part of the duplex name. The duplex name can comprise a suffix (number following the decimal point in a duplex name) that merely refers to a batch production number. The suffix can be omitted from the duplex name without changing the chemical structure. For example, duplex AD-953481.1 in Table 4A refers to the same duplex as AD-953481 in Table 13. Duplex Name Strand Target Modified Sequence (5′-3′) SEQ ID NO AD-64958 sense None asascaguGfuUfCfUfugcucuauaaL96 5003 anti- None usUfsauaGfagcaagaAfcAfcuguususu 5004 sense AD-953481 sense VEGF-A asgsugc(Uhd)aadTgdTuauugguguaL96 1038 anti- VEGF-A VPusdAscadCcdAauaadCadTudAgcacusgsu 1168 sense AD-901349 sense VEGF-A asasgac(Uhd)GfaUfAfCfagaacgaucaL96 4156 anti- VEGF-A VPusGfsaucg(Tgn)ucuguaUfcAfgucuususc  130 sense AD-953338 sense VEGF-A asgsacu(Ghd)AfuAfCfAfgaacgaucgaL96  520 anti- VEGF-A VPusCfsgauCfgUfUfcuguAfuCfagucususu  650 sense AD-953341 sense VEGF-A csusgau(Ahd)CfaGfAfAfcgaucgauaaL96  532 anti- VEGF-A VPusUfsaucGfaUfCfguucUfgUfaucagsusc  662 sense AD-901355 sense VEGF-A csgsaca(Ghd)AfaCfAfGfuccuuaaucaL96    4 anti- VEGF-A VPusGfsauua(Agn)ggacugUfuCfugucgsasu  133 sense AD-901356 sense VEGF-A csasgaa(Chd)AfgUfCfCfuuaauccagaL96    3 anti- VEGF-A VPusCfsugga(Tgn)uaaggaCfuGfuucugsusc  132 sense AD-953350 sense VEGF-A asascag(Uhd)GfcUfAfAfuguuauuggaL96  524 anti- VEGF-A VPusCfscaaUfaAfCfauuaGfcAfcuguusasa  654 sense AD-953365 sense VEGF-A csgsgua(Chd)UfuAfUfUfuaauaucccaL96  552 anti- VEGF-A VPusGfsggaUfaUfUfaaauAfaGfuaccgsusa  682 sense AD-953370 sense VEGF-A gscsucu(Chd)UfuAfUfUfuguaccgguaL96  533 anti- VEGF-A VPusAfsccgGfuAfCfaaauAfaGfagagcsasa  663 sense AD-953375 sense VEGF-A asasaua(Ghd)AfcAfUfUfgcuauucugaL96  530 anti- VEGF-A VPusCfsagaAfuAfGfcaauGfuCfuauuususa  660 sense AD-953386 sense VEGF-A csgsaag(Uhd)GfgUfGfAfaguucauggaL96  541 anti- VEGF-A VPusCfscauGfaAfCfuucaCfcAfcuucgsusg  671 sense AD-953410 sense VEGF-A gsasaag(Uhd)GfuUfUfUfauauacgguaL96  585 anti- VEGF-A VPusAfsccgUfaUfAfuaaaAfcAfcuuucsusc  715 sense AD-953411 sense VEGF-A gsusuuu(Ahd)UfaUfAfCfgguacuuauaL96  584 anti- VEGF-A VPusAfsuaaGfuAfCfcguaUfaUfaaaacsasc  714 sense

TABLE 14 VEGF-A in vivo single-dose screen with one set of exemplary VEGF-A siRNA duplexes. In this table, the columns “Duplex Name” and “Strand Name” provide the numerical part of the duplex or strand name. The duplex or strand name can comprise a suffix (number following the decimal point in a duplex name) that merely refers to a batch production number. The suffix can be omitted from the duplex name without changing the chemical structure. For example, the antisense strand name A-2521293.1 in Table 10A refers to the same antisense strand as A-2521293 in Table 14. SEQ Unmodified SEQID NO Duplex Strand ID NO Modified Sequence Sequence (un- Name Name Strand Target (modified) (5′-3′) (5′-3′) modified) AD- A- sense None 5003 asascaguGfuUfCfUfu AACAGUGUUC 5021 64958 128009 gcucuauaaL96 UUGCUCUAUA A A- anti- None 5004 usUfsauaGfagcaaga UUAUAGAGCA 5022 126312 sense AfcAfcuguususu AGAACACUGU UUU AD- A- sense VEGF- 1044 asasgac(Uhd)gadTad AAGACUGATAC 1304 1397068 1700995 A CagaacgaucaL96 AGAACGAUCA A- anti- VEGF- 3901 VPusdGsaudCg(U2p) UGAUCGUUCU 4081 2521293 sense A ucugdTadTcdAgucu GTATCAGUCU USUSC UUC AD- A- sense VEGF- 10 csusgau(Ahd)CfaGfA CUGAUACAGA 268 1397052 1701263 A fAfcgaucgauaaL96 ACGAUCGAUA A A- anti- VEGF- 3957 VPusUfsaudCg(A2p) UUAUCGAUCG 4137 2521192 sense A ucguucUfgUfaucags UUCUGUAUCA use GUC AD- A- sense VEGF- 5005 asusgcagAfuUfAfUfg AUGCAGAUUA 5023 1397050 2600337 A cgg(Ahd)ucaaaL96 UGCGGAUCAA A A- anti- VEGF- 3936 VPusUfsugdAu(C2p) UUUGAUCCGC 4116 2521186 sense A cgcauaAfuCfugcausg AUAAUCUGCA sg UGG AD- A- sense VEGF- 5006 ascscaggAfaAfGfAfc ACCAGGAAAG 5024 1397051 2600338 A uga(Uhd)acagaL96 ACUGAUACAG A A- anti- VEGF- 3918 VPusCfsuguAfucagu UCUGUAUCAG 4098 2521190 sense A cuUfuCfcuggusgsc UCUUUCCUGG UGC AD- A- sense VEGF- 5007 asgsaac(Ahd)GfuCfC AGAACAGUCC 5025 1397053 2600339 A fUfuaauccagaaL96 UUAAUCCAGA A A- anti- VEGF- 3924 VPusUfscudGg(A2p) UUCUGGAUUA 4104 2521200 sense A uuaaggAfcUfguucus AGGACUGUUC gsu UGU AD- A- sense VEGF- 5008 asgsauu(Ahd)GfaGfA AGAUUAGAGA 5026 1397054 2600340 A fGfuuuuauuucaL96 GUUUUAUUUC A A- anti- VEGF- 2640 VPusGfsaaaUfaaaac UGAAAUAAAA 4110 2282496 sense A ucUfcUfaaucususc CUCUCUAAUC UUC AD- A- sense VEGF- 5009 asasaag(Ahd)GfaAfA AAAAGAGAAA 5027 1397055 2600341 A fGfuguuuuauaaL96 GUGUUUUAUA A A- anti- VEGF- 2775 VPusUfsauaAfaacac UUAUAAAACA 3673 2282766 sense A uuUfcUfcuuuuscsu CUUUCUCUUU UCU AD- A- sense VEGF- 5010 asasagagAfaAfGfUfg AAAGAGAAAG 5028 1397056 2600342 A uuu(Uhd)auauaL96 UGUUUUAUAU A A- anti- VEGF- 2776 VPusAfsuauAfaaaca UAUAUAAAAC 3674 2282768 sense A cuUfuCfucuuususc ACUUUCUCUU UUC AD- A- sense VEGF- 5011 csusacagcaCfAfAfca CUACAGCACAA 5029 1397058 2600344 A aa(Uhd)gugaaL96 CAAAUGUGAA A- anti- VEGF- 3953 VPusdTscadCadTuug UTCACATUUG 4133 2521229 sense A udTgUfgcuguagsgsg UTGUGCUGUA GGG AD- A- sense VEGF- 5012 asasaga(Chd)ugAfUf AAAGACUGAU 5030 1397059 2600345 A AfcagaacgauaL96 ACAGAACGAU A A- anti- VEGF- 3889 VPusdAsucdGudTcu UAUCGUTCUG 4069 2521233 sense A gudAuCfagucuuuscs UAUCAGUCUU c UCC AD- A- sense VEGF- 5013 asasgac(Uhd)gaUfAf AAGACUGAUA 5031 1397060 2600346 A CfagaacgaucaL96 CAGAACGAUC A A- anti- VEGF- 3902 VPusdGsaudCg(U2p) UGAUCGUUCU 5039 2521235 sense A ucugdTaUfcagucuus GTAUCAGUCU use UUC AD- A- sense VEGF- 5014 asusacagaaCfGfAfuc AUACAGAACG 5032 1397061 2600347 A ga(Uhd)acagaL96 AUCGAUACAG A A- anti- VEGF- 3932 VPusdCsugdTa(U2p) UCUGTAUCGA 4112 2521239 sense A cgaudCgUfucuguaus UCGUUCUGUA esg UCG AD- A- sense VEGF- 5015 csasgaa(Chd)agUfCf CAGAACAGUCC 5033 1397062 2600348 A CfuuaauccagaL96 UUAAUCCAGA A- anti- VEGF- 3944 VPusdCsugdGa(U2p) UCUGGAUUAA 4124 2521245 sense A uaagdGaCfuguucugs GGACUGUUCU use GUC AD- A- sense VEGF- 5016 asusugg(Ahd)uuCfGf AUUGGAUUCG 5034 1397064 2600350 A CfcauuuuauuaL96 CCAUUUUAUU A A- anti- VEGF- 3938 VPusdAsaudAadAau UAAUAAAAUG 4118 2521257 sense A ggdCgAfauccaaususc GCGAAUCCAA UUC AD- A- sense VEGF- 5017 gsasuucgccAfUfUfuu GAUUCGCCAU 5035 1397065 2600351 A au(Uhd)uuucaL96 UUUAUUUUUC A A- anti- VEGF- 3965 VPusdGsaadAadAua UGAAAAAUAA 4145 2521259 sense A aadAuGfgcgaaucscs AAUGGCGAAU g CCG AD- A- sense VEGF- 5018 gsasgaa(Ahd)guGfUf GAGAAAGUGU 5036 1397066 2600352 A UfuuauauacgaL96 UUUAUAUACG A A- anti- VEGF- 3962 VPusdCsgudAudAua UCGUAUAUAA 4142 2521271 sense A aadAcAfcuuucucsus AACACUUUCU u CUU AD- A- sense VEGF- 5019 gsusguu(Uhd)uaUfA GUGUUUUAUA 5037 1397067 2600353 A fUfacgguacuuaL96 UACGGUACUU A A- anti- VEGF- 3971 VPusd AsagdT adCcgu UAAGTACCGU 4151 2521275 sense A adTaUfaaaacacsusu ATAUAAAACAC UU AD- A- sense VEGF- 5020 asgsauu(Ahd)gadGa AGAUUAGAGA 5038 1397069 2600354 A dGuuuuauuucaL96 GUUUUAUUUC A A- anti- VEGF- 3928 VPusdGsaadAudAaa UGAAAUAAAA 4108 2521319 sense A acdTcdTcdTaaucusu CTCTCTAAUCU sc UC

Experimental Methods

An AAV vector harboring Homo sapiens VEGF-A was injected in 6-8 week old C57BL/6 female mice (2×10¹¹ viral particles/mouse), and at 14 days post-AAV administration, a selected siRNA or a control agent were subcutaneously injected at 3 mg/kg in mice (n=3 per group). Mice were sacrificed and their livers were assessed for VEGF-A mRNA levels at 14 days post-injection of the siRNAs or control.

Results

Table 15 and FIG. 2 demonstrate the results of the in vivo screen with the siRNA duplexes corresponding to the siRNA sequences in Table 12. Of the siRNA duplexes evaluated in vivo in Table 15, 2 achieved a knockdown of VEGF-A of ≥60%, 4 achieved a knockdown of VEGF-A of ≥50%, 9 achieved a knockdown of VEGF-A of ≥40%, 11 achieved a knockdown of VEGF-A of ≥30%, and 13 achieved a knockdown of VEGF-A of ≥15%.

TABLE 15 Efficacy of exemplary VEGF-A siRNAs in mice. In this table the column “Duplex Name” provides the numerical part of the duplex name with a suffix (number following the decimal point in a duplex name) that merely refers to a batch production number. The suffix can be omitted from the duplex name without changing the chemical structure. For example, duplex AD-953504 in Table 12 refers to the same duplex as AD-953504.2 in Table 15. Day 14 post-treatment Duplex % VEGF-A (Administered at 3 mg/kg) Message Remaining St Dev PBS 101.85 21.59 Naïve 106.47 14.34 AD-64228.39 62.99 16.53 AD-901376.2 72.86 10.62 AD-953308.2 81.89 34.49 AD-953336.2 51.31 16.11 AD-953337.2 44.93  5.57 AD-953339.2 58.33 25.29 AD-953340.2 33.05 18.66 AD-953342.2 35.97  8.19 AD-953344.2 56.71 14.45 AD-953351.2 43.75 29.11 AD-953363.2 52.28 10.56 AD-953364.2 69.25 10.87 AD-953374.2 59.51 18.65 AD-953504.2 63.66 10.61

Table 16 and FIG. 4 demonstrate the results of the in vivo screen with the siRNA duplexes corresponding to the siRNA sequences in Table 13. Of the siRNA duplexes evaluated in vivo in Table 16, 3 achieved a knockdown of VEGF-A of ≥70%, 6 achieved a knockdown of VEGF-A of ≥60%, 9 achieved a knockdown of VEGF-A of ≥50%, 12 achieved a knockdown of VEGF-A of ≥40%, and 13 achieved a knockdown of VEGF-A of ≥30%.

TABLE 16 Efficacy of exemplary VEGF-A siRNAs in mice. In this table the column “Duplex Name” provides the numerical part of the duplex name with a suffix (number following the decimal point in a duplex name) that merely refers to a batch production number. The suffix can be omitted from the duplex name without changing the chemical structure. For example, duplex AD-901349 in Table 13 refers to the same duplex as AD-901349.1 in Table 16. Day 14 post-treatment Duplex % VEGF-A (Administered at 3 mg/kg) Message Remaining St Dev PBS 102.2 24.0 Naïve 56.8  8.9 AD-901349.1 26.4 14.1 AD-953481.1 22.6 26.2 AD-901356.1 34.5  6.4 AD-901355.1 43.0  4.4 AD-953365.1 60.5  3.3 AD-953410.1 38.8 16.4 AD-953411.1 56.2 10.5 AD-953338.1 58.1  5.6 AD-953350.1 42.0  2.5 AD-953375.1 45.6  6.1 AD-953341.1 30.1 12.4 AD-953370.1 28.3  8.5 AD-953386.1 52.9 13.3 AD-64958 57.1  6.1 (ELF8 TTR control)

Table 17 and FIG. 6 demonstrate the results of the in vivo screen with the siRNA duplexes corresponding to the siRNA sequences in Table 14. Of the siRNA duplexes evaluated in vivo in Table 17, 5 achieved a knockdown of VEGF-A of ≥40%, 10 achieved a knockdown of VEGF-A of ≥30%, 15 achieved a knockdown of VEGF-A of ≥20%, and 17 achieved a knockdown of VEGF-A of ≥10%.

TABLE 17 Efficacy of exemplary VEGF-A siRNAs in mice. In this table the column “Duplex Name” provides the numerical part of the duplex name with a suffix (number following the decimal point in a duplex name) that merely refers to a batch production number. The suffix can be omitted from the duplex name without changing the chemical structure. For example, duplex AD-1397050 in Table 14 refers to the same duplex as AD-1397050.2 in Table 17. Day 14 post-treatment Duplex % VEGF-A (Administered at 3 mg/kg) Message Remaining St Dev PBS 89.7 45.3 Naïve 100.0 17.4 AD-1397050.2 50.1 15.3 AD-1397051.2 76.7 28.9 AD-1397052.2 63.6 19.6 AD-1397053.2 50.9 15.6 AD-1397054.2 53.0 12.1 AD-1397055.2 84.4 32.4 AD-1397056.2 59.2 23.8 AD-1397058.2 77.5 20.7 AD-1397059.2 77.1 13.4 AD-1397060.2 68.9  6.8 AD-1397061.2 58.2 12.9 AD-1397062.2 68.7 12.0 AD-1397064.2 62.9  6.7 AD-1397065.2 85.2 29.9 AD-1397066.2 77.7  8.7 AD-1397067.2 93.5 37.3 AD-1397068.2 62.0 15.7 AD-1397069.2 76.9 26.1 AD-64958.100 66.0  2.6 

We claim:
 1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of vascular endothelial growth factor A (VEGF-A), wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A, and 18B, and wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 8A, 8B, 10A, 10B, 12, 13, 14, 18A, and 18B that corresponds to the antisense sequence.
 2. The dsRNA agent of claim 1, wherein the portion of the sense strand is a portion within nucleotides 1855-1875, 1858-1878, 2178-2198, 2181-2201, 2944-2964, 2946-2966, 2952-2972, 3361-3381, or 3362-3382 of SEQ ID NO:
 1. 3. The dsRNA agent of claim 1 or 2, wherein the portion of the sense strand is a portion within a sense strand from a duplex chosen from AD-1020574 (CGACAGAACAGUCCUUAAUCA (SEQ ID NO: 4200)), AD-901094 (CAGAACAGUCCUUAAUCCAGA (SEQ ID NO: 4201)), AD-1020575 (CAGAACAGUCCUUAAUCCAGA (SEQ ID NO: 4202)), AD-901100 (AACAGUGCUAAUGUUAUUGGA (SEQ ID NO: 4203)), AD-901101 (AGUGCUAAUGUUAUUGGUGUA (SEQ ID NO: 4204)), AD-901113 (GAGAAAGUGUUUUAUAUACGA (SEQ ID NO: 4205)), AD-901123 (AAAAUAGACAUUGCUAUUCUA (SEQ ID NO: 4206)), AD-901124 (AAAUAGACAUUGCUAUUCUGA (SEQ ID NO: 4207)), AD-901158 (GAAAGUGUUUUAUAUACGGUA (SEQ ID NO: 4208)), AD-901159 (GUUUUAUAUACGGUACUUAUA (SEQ ID NO: 4209)), AD-1020573 (AGUGCUAATGTUAUUGGUGUA (SEQ ID NO: 4210)), or AD-1023143 (AAAAUAGACATUGCUAUUCUA (SEQ ID NO: 4211)).
 4. The dsRNA agent of any one of claims 1-3, wherein the portion of the sense strand is a sense strand chosen from the sense strands of AD-1020574 (CGACAGAACAGUCCUUAAUCA (SEQ ID NO: 4200)), AD-901094 (CAGAACAGUCCUUAAUCCAGA (SEQ ID NO: 4201)), AD-1020575 (CAGAACAGUCCUUAAUCCAGA (SEQ ID NO: 4202)), AD-901100 (AACAGUGCUAAUGUUAUUGGA (SEQ ID NO: 4203)), AD-901101 (AGUGCUAAUGUUAUUGGUGUA (SEQ ID NO: 4204)), AD-901113 (GAGAAAGUGUUUUAUAUACGA (SEQ ID NO: 4205)), AD-901123 (AAAAUAGACAUUGCUAUUCUA (SEQ ID NO: 4206)), AD-901124 (AAAUAGACAUUGCUAUUCUGA (SEQ ID NO: 4207)), AD-901158 (GAAAGUGUUUUAUAUACGGUA (SEQ ID NO: 4208)), AD-901159 (GUUUUAUAUACGGUACUUAUA (SEQ ID NO: 4209)), AD-1020573 (AGUGCUAATGTUAUUGGUGUA (SEQ ID NO: 4210)), or AD-1023143 (AAAAUAGACATUGCUAUUCUA (SEQ ID NO: 4211)).
 5. The dsRNA of any one of claims 1-4, wherein the portion of the antisense strand is a portion within an antisense strand from a duplex chosen from AD-1020574 (UGAUUAAGGACUGUUCUGUCGAU (SEQ ID NO: 4212)), AD-901094 (UCUGGAUUAAGGACUGUUCUGUC (SEQ ID NO: 4213)), AD-1020575 (UCUGGATUAAGGACUGUUCUGUC (SEQ ID NO: 4214)), AD-901100 (UCCAAUAACAUUAGCACUGUUAA (SEQ ID NO: 4215)), AD-901101 (UACACCAAUAACAUUAGCACUGU (SEQ ID NO: 4216)), AD-901113 (UCGUAUAUAAAACACUUUCUCUU (SEQ ID NO: 4217)), AD-901123 (UAGAAUAGCAAUGUCUAUUUUAU (SEQ ID NO: 4218)), AD-901124 (UCAGAAUAGCAAUGUCUAUUUUA (SEQ ID NO: 4219)), AD-901158 (UACCGUAUAUAAAACACUUUCUC (SEQ ID NO: 4220)), AD-901159 (UAUAAGUACCGUAUAUAAAACAC (SEQ ID NO: 4221)), AD-1020573 (UACACCAAUAACATUAGCACUGU (SEQ ID NO: 4222)), or AD-1023143 (UAGAAUAGCAATGTCTAUUUUAU (SEQ ID NO: 4223)).
 6. The dsRNA of any one of claims 1-5, wherein the portion of the antisense strand is an antisense strand chosen the antisense strands of AD-1020574 (UGAUUAAGGACUGUUCUGUCGAU (SEQ ID NO: 4212)), AD-901094 (UCUGGAUUAAGGACUGUUCUGUC (SEQ ID NO: 4213)), AD-1020575 (UCUGGATUAAGGACUGUUCUGUC (SEQ ID NO: 4214)), AD-901100 (UCCAAUAACAUUAGCACUGUUAA (SEQ ID NO: 4215)), AD-901101 (UACACCAAUAACAUUAGCACUGU (SEQ ID NO: 4216)), AD-901113 (UCGUAUAUAAAACACUUUCUCUU (SEQ ID NO: 4217)), AD-901123 (UAGAAUAGCAAUGUCUAUUUUAU (SEQ ID NO: 4218)), AD-901124 (UCAGAAUAGCAAUGUCUAUUUUA (SEQ ID NO: 4219)), AD-901158 (UACCGUAUAUAAAACACUUUCUC (SEQ ID NO: 4220)), AD-901159 (UAUAAGUACCGUAUAUAAAACAC (SEQ ID NO: 4221)), AD-1020573 (UACACCAAUAACATUAGCACUGU (SEQ ID NO: 4222)), or AD-1023143 (UAGAAUAGCAATGTCTAUUUUAU (SEQ ID NO: 4223)).
 7. The dsRNA of any one of claims 1-6, wherein the sense strand and the antisense strand comprise nucleotide sequences of the paired sense strand and antisense strand of a duplex selected from AD-1020574 (SEQ ID NO: 4200 and 4212), AD-901094 (SEQ ID NO: 4201 and 4213), AD-1020575 (SEQ ID NO: 4202 and 4214), AD-901100 (SEQ ID NO: 4203 and 4215), AD-901101 (SEQ ID NO: 4204 and 4216), AD-901113 (SEQ ID NO: 4205 and 4217), AD-901123 (SEQ ID NO: 4206 and 4218), AD-901124 (SEQ ID NO: 4207 and 4219), AD-901158 (SEQ ID NO: 4208 and 4220), AD-901159 (SEQ ID NO: 4209 and 4221), AD-1020573 (SEQ ID NO: 4210 and 4222), or AD-1023143 (SEQ ID NO: 4211 and 4223).
 8. The dsRNA agent of any one of claims 1-7, wherein the antisense strand comprises a nucleotide sequence of an antisense sequence listed in Table 18A, and the sense strand comprises a nucleotide sequence of a sense sequence listed in Table 18A that corresponds to the antisense sequence.
 9. The dsRNA agent of any one of claims 1-8, wherein the dsRNA agent is AD-1020574, AD-901094, AD-1020575, AD-901100, AD-901101, AD-901113, AD-901123, AD-901124, AD-901158, AD-901159, AD-1020573, or AD-1023143.
 10. The dsRNA agent of any one of claims 1-9, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties.
 11. The dsRNA agent of claim 10, wherein the lipophilic moiety is conjugated via a linker or carrier.
 12. The dsRNA agent of claim 10 or 11, wherein one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand.
 13. The dsRNA agent of claim 12, wherein the one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand via a linker or carrier.
 14. The dsRNA agent of any one of claims 10-13, wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.
 15. The dsRNA agent of claim 14, wherein the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.
 16. The dsRNA agent of any one of claims 10-15, wherein the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) or the double stranded region.
 17. The dsRNA agent of any one of claims 10-15, wherein the lipophilic moiety is conjugated to the sense strand or the antisense strand via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.
 18. The dsRNA agent of any one of claims 10-16, wherein the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.
 19. The dsRNA agent of any of the preceding claims, wherein the dsRNA agent comprises at least one modified nucleotide.
 20. The dsRNA agent of claim 19, wherein no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand are unmodified nucleotides.
 21. The dsRNA agent of claim 19, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.
 22. The dsRNA agent of any one of claims 19-21, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′-phosphate mimic, a glycol modified nucleotide, and a 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof.
 23. The dsRNA agent of any of the preceding claims, wherein at least one strand comprises a 3′ overhang of at least 2 nucleotides.
 24. The dsRNA agent of any of the preceding claims, wherein the double stranded region is 15-30 nucleotide pairs in length.
 25. The dsRNA agent of claim 24, wherein the double stranded region is 17-23 nucleotide pairs in length.
 26. The dsRNA agent of any of the preceding claims, wherein each strand has 19-30 nucleotides.
 27. The dsRNA agent of any of the preceding claims, wherein the agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.
 28. The dsRNA agent of any one of claims 10-27, further comprising a targeting ligand, e.g., a ligand that targets an ocular tissue.
 29. The dsRNA agent of claim 28, wherein the ocular tissue is a retinal pigment epithelium (RPE) or choroid tissue, e.g., a choroid vessel.
 30. The dsRNA agent of any one of the preceding claims, further comprising a phosphate or phosphate mimic at the 5′-end of the antisense strand.
 31. The dsRNA agent of claim 30, wherein the phosphate mimic is a 5′-vinyl phosphonate (VP).
 32. The dsRNA agent of any one of the preceding claims, comprising: (i) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 4164, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4176; (ii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1465, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4177; (iii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1466, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4178; (iv) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1467, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4179; (v) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1468, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4180; (vi) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1469, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4181; (vii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1470, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4182; (viii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1471, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4183; (ix) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1472, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4184; (x) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1473, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4185; (xi) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1474, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4186; or (xii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 1475, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO:
 4187. 33. A cell containing the dsRNA agent of any one of claims 1-32.
 34. A pharmaceutical composition for inhibiting expression of a VEGF-A, comprising the dsRNA agent of any one of claims 1-32.
 35. A method of inhibiting expression of VEGF-A in a cell, the method comprising: (a) contacting the cell with the dsRNA agent of any one of claims 1-32, or a pharmaceutical composition of claim 34; and (b) maintaining the cell produced in step (a) for a time sufficient to reduce levels of VEGF-A mRNA, VEGF-A protein, or both of VEGF-A mRNA and protein, thereby inhibiting expression of VEGF-A in the cell.
 36. The method of claim 35, wherein the cell is within a subject.
 37. The method of claim 36, wherein the subject is a human.
 38. The method of claim 37, wherein the subject has been diagnosed with a VEGF-A-associated disorder, e.g., wet age-related macular degeneration (wet AMD), diabetic retinopathy (DR), diabetic macular edema (DME), retinal vein occlusion (RVO), macular edema following retinal vein occlusion (MEfRVO), retinopathy of prematurity (ROP), or myopic choroidal neovascularization (mCNV).
 39. A method of treating a subject diagnosed with a VEGF-A-associated disorder comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1-23 or a pharmaceutical composition of claim 25, thereby treating the disorder.
 40. The method of claim 39, wherein the VEGF-A-associated disorder is an angiogenic ocular disorder.
 41. The method of claim 40, wherein the angiogenic ocular disorder is selected from the group consisting of AMD, DR, DME, RVO, MEfRVO, ROP, and mCNV.
 42. The method of any one of claims 39-41, wherein treating comprises amelioration of at least one sign or symptom of the disorder.
 43. The method of any one of claims 39-42, wherein the treating comprises (a) inhibiting angiogenesis; (b) inhibiting or reducing the expression or activity of VEGF-A; (c) inhibiting choroidal neovascularization; (d) inhibiting growth of new blood vessels in the choriocapillaris; (e) reducing retinal thickness; (f) increasing visual acuity; or (g) reducing intraocular inflammation.
 44. The method of any one of claims 36-43, wherein the dsRNA agent is administered to the subject intraocularly, intravenously, or topically.
 45. The method of claim 44, wherein the intraocular administration comprises intravitreal administration (e.g., intravitreal injection), transscleral administration (e.g., transscleral injection), subconjunctival administration (e.g., subconjunctival injection), retrobulbar administration (e.g., retrobulbar injection), intracameral administration (e.g., intracameral injection), or subretinal administration (e.g., subretinal injection).
 46. The method of any one of claims 36-45, further comprising administering to the subject an additional agent or therapy suitable for treatment or prevention of an VEGF-A-associated disorder (e.g., one or more of a photodynamic therapy, photocoagulation therapy, a steroid, a non-steroidal anti-inflammatory agent, an anti-VEGF agent, or a vitrectomy). 